Evidence that elevated intracellular cyclic AMP triggers spore maturation in Dictyostelium
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Development 105, 753-759 (1989) 753
Printed in Great Britain © The Company of Biologists Limited 1989
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 in differentiation in wild-type amoebae incubated in sub-
Dictyostelium when environmental influences induce a merged monolayers. These analogues also stimulate
migrating slug to transform into a fruiting body. As the accumulation of UDP-galactose epimerase in slug cells
amoeboid prespore cells turn into refractile spores there transferred to shaken suspension.
is a burst of enzyme accumulation, including UDP- The ability to induce spore differentiation with Br-
galactose epimerase, and at a later stage the exocytosis of cAMP in wild-type strains provides a new technique that
preformed components of the spore coat. Evidence is can be exploited in various ways. For instance, spore
presented here that this process is triggered by an differentiation in strain V12M2 is induced by 8-Br-
elevated intracellular cAMP concentration. cAMP at very low cell densities, suggesting that neither
First, a number of rapidly developing (rde) mutants, cell contact nor additional soluble inducers are necessary
whose cAMP metabolism had been investigated pre- in these conditions. In contrast NC4 cells may require an
viously, are shown to be able to form spores in sub- additional inducer. Spore differentiation is inhibited by
merged monolayers, whereas wild-type strains are not. the stalk- specific inducer DIF-1 suggesting that DIF-1
The phenotypes of these mutants are best explained by a inhibits a target downstream of intracellular cAMP in
derepression of the signal transduction pathway utilizing the signal transduction pathway inducing spore differen-
intracellular cAMP. tiation.
Second and more direct, it is shown that the permeant
cAMP analogues 8-Br-cAMP and 8-chlorophenylthio- Key words: Dictyostelium, spore maturation, intracellular
cAMP, but not cAMP itself, can rapidly induce spore cAMP, 8-bromo-cyclic AMP.
Introduction aggregate (Newell et al. 1969; Schindler & Sussman,
1977). In addition the slugs appear to accumulate a low
The migrating slug of Dictyostelium discoideum is an Mr metabolite, possibly a weak acid, that is necessary
arrested stage of development which can persist for for culmination (Sussman et al. 1978). Presumably these
days before it is triggered by suitable environmental influences must be transduced into the individual cells
conditions to transform into a mature fruiting body. As within the slug and integrated to trigger changes in both
this transformation proceeds, there is a radical reorgan- gene expression and cellular morphogenesis.
ization of the slug, accompanied by the overt differen- The initial differentiation of prespore cells appears to
tiation of stalk and spore cells from their amoeboid be induced by extracellular cAMP (Kay et al. 1978; Kay,
precursors. The prestalk cells sequentially vacuolate 1979) and, since there is evidence that cAMP signalling
and lay down cellulose as they move into the growing persists to later stages of development (Bonner, 1949;
tip of the stalk. The prespore cells are carried aloft by Nestle & Sussman, 1972; Schaap & Wang, 1984), some
the stalk and transform synchronously into refractile- modulation of cAMP signalling may be involved in
walled spores in a process that involves a burst of triggering spore maturation. cAMP signals are trans-
accumulation of enzymes such as UDP-galactose epi- duced into at least three intracellular second messen-
merase (Newell & Sussman, 1970) and, at a later stage, gers: cAMP itself, cGMP and inositol phosphates
the exocytosis of preformed components of the spore (Gerisch, 1987; Europe-Finner & Newell, 1987). How-
coat (Hohl & Hamamoto, 1969; Maeda & Takeuchi, ever, the individual roles of the second messengers in
1969). producing changes in gene expression, cell movement
The transformation of a slug into a fruiting body and morphogenesis have not yet been clearly dis-
(culmination) can be triggered by overhead light, a tinguished.
drop in humidity or the loss of ammonia from the The hypothesis will be advanced here that spore754 R. R. Kay
maturation is triggered by elevated levels of intracellu- Table 1.
lar cAMP. This idea was first suggested by the pheno- % cell type
types of certain mutants facilitated in spore maturation
and then powerfully supported by the use of permeant Strain Stalk Spore
cAMP analogues to induce spore formation in wild-type V12M2 (parent) 56-7 0
strains. The techniques for spore induction then al- HM15 (sporogenous) 57-0 34-5
lowed further questions to be asked about the possible NC4 (parent) 0 0
role of signals, such as cell-cell contact, in wild-type Frl7 (rdeA) 5-6 66-8
spore differentiation. HTY507 (rdeA) 1-0 40-3
HTY509 (rdeA) 1-6 42-9
HTY506 (rdeC) 0 43-0
HTY217 (rdeC) 0 65-1
Materials and methods
Rapidly developing (rde) mutants are sporogenous (differentiate
8-(4-chlorophenylthio)-cyclic AMP was from Boehringer, into spores in submerged monolayers in the presence of cAMP).
cAMP (cAMP), 2'-deoxy-cyclic-AMP and 8-bromo-cAMP The ultimate parent of the rde mutants is NC4 and this strain is
(Br-cAMP) were from Sigma. Br-cAMP was also synthesized included for comparison, as is an authentic sporogenous mutant,
by bromination of cAMP with bromine water (Muneyama et HM15, and its ultimate parent V12M2. Monolayers of cells of each
genotype were submerged at lO'cm" 2 in a simple salts medium
al. 1971). The precipitate was purified by first dissolving in
supplemented with 5 IHM-CAMP (see Materials and methods). After
water by bringing to pH~8 with KOH and then reprecipitat- 48 h stalk and spore cell differentiation was scored microscopically
ing with HC1. The product remained pale brown after 3 cycles and the results given as a percentage of total cells. Results are
of purification but did not contain detectable impurities on means of 2-6 separate plates
TLC (Muneyama et al. 1971, system B) and behaved identi-
cally with the commercial material in the experiments to be
described. DIF-1 was synthesized as described (Masento et al.
1988), and synthetic discadenine was a kind gift from Dr Y. (Town et al. 1976; Kay et al. 1978). These mutants are
Tanaka (Abe et al. 1976). therefore considered to be facilitated in the maturation
The sporogenous mutant HM15 derives ultimately from
of prespore cells into spores. The rapidly developing
strain V12M2 and was selected from its immediate parent, (rde) mutants were isolated because they form spores
HM2, by virtue of its ability to form detergent-resistant spores prematurely in normal development (Sonneborn et al.
when incubated in a monolayer with cAMP (Town etal. 1976; 1963; Kessin, 1977; Abe & Yanagisawa, 1983) and were
Kay et al. 1978; Kay, 1987). The rapidly developing mutants of interest because their lesion had been linked to an
HTY 217, HTY 506, HTY 507 and HTY 509 were a kind gift altered intracellular cAMP metabolism (see below) and
from Drs K. Abe and K. Yanagisawa (Abe & Yanagisawa, because of their phenotypic resemblance to some of the
1983) and Frl7 (Sonneborn et al. 1963) from Dr C. D. Town. sporogenous mutants. For instance, in conditions suit-
Cells were grown on Klebsiella aerogenes and prepared for able for normal development, both the sporogenous
development as previously described (Kay, 1987). Slugs were
obtained by allowing cells to develop on 1-8% L28 agar mutant HM15 and the rdeC mutants arrest as mounds
(Oxoid) containing 20 mM-KCl, 20mM-NaCl, 1 mM-CaCl2 and and produce spores several hours earlier than their
after 18 h harvested, partially disaggregated by syringing respective parents. Similarly HM18 and the rdeA mu-
through a 19-gauge needle and resuspended at a nominal cell tants arrest as early culminates and spore differen-
density of 2x10^ cells ml"1. Suspensions were shaken in tiation is again premature. These similarities suggested
conical flasks at 180 revs min"1. The medium for develop- that some of the rde and sporogenous mutants might be
ment, in suspension or in monolayers in Sterilin tissue culture allelic. Unfortunately a direct genetic test of this idea is
dishes, was 10mM-2-(N-morpholino)-ethanesulphonic acid, difficult, because the two groups of mutants were
20 mM-KCl, 20mM-NaCl, lmM-MgCl2, lmM-CaCl2 pH6-2
containing 15/igmP 1 tetracycline, 200/igml"1 streptomycin isolated in the V12 and NC4 backgrounds, which are
sulphate and cyclic nucleotides as indicated ('spore medium', incompatible in parasexual crosses (Robson & Wil-
Kay, 1982, 1987). Cell differentiation was monitored by liams, 1979). However, it has already been shown that
phase-contrast microscopy. the rdeA mutant Frl7 is sporogenous (Town et al. 1976)
UDP-galactose-4-epimerase (EC 5.1.3.2) was assayed by a and Table 1 shows that all rde mutants of both available
coupled spectrophotometric assay (Telser & Sussman, 1971) complementation groups (rdeB is lost) are sporogen-
at 35°C and protein by a dye-binding assay (Bradford, 1976). ous, that is they make spores in monolayers when
incubated with cAMP. Kessin (1977) suggested that the
rde phenotype might be due to an overproduction of
Results intracellular cAMP, which in turn acted as an inducer of
developmental gene expression. Altered cAMP metab-
Mutants in spore maturation olism in the rde mutants was subsequently confirmed by
The initial clue linking intracellular cAMP to spore direct measurement (Coukell & Chan, 1980; Abe &
maturation came from the phenotypes of two sets of Yanagisawa, 1983). Rde A mutants have elevated
independently isolated mutants in which spore matu- intracellular cAMP levels as expected, but surprisingly
ration is facilitated or 'derepressed'. The sporogenous rdeC mutants have very low levels. However, this
mutants were isolated because they are able to form paradoxical property of rdeC mutants can be explained
spores in submerged monolayers with cAMP, whereas within the original hypothesis, since in both yeast and
their parents arrest as amoeboid prespore cells and mammalian cells cAMP levels are controlled by nega-
never (Induction of spore maturation by cAMP in Dictyostelium 755
protein kinase (Nikawa et al. 1987; Gettys et al. 1987). necessary with mammalian cells but higher concen-
Thus a constitutively active protein kinase would feed trations were also explored with Dictyostelium cells,
back to inhibit adenyl cyclase and produce low cAMP because of their relative impermeability.
levels, as seen in the rdeC mutants, while producing the It is apparent from Fig. 1 that high concentrations of
downstream effects of elevated cAMP levels. Br-cAMP can induce greater than 70% spore forma-
The results with the spore maturation mutants tion amongst amoebae of strain V12M2 incubated from
suggest that elevated intracellular levels of cAMP the start of development with the inducer in submerged
induce spore maturation, but a more direct test of this monolayers. In these experiments, spore formation
idea was required. started after about 16 h. Spores could also be induced in
strain NC4 though less efficiently (see later). The
induced spores stain with a spore-specific antibody
Spore induction in wild-type strains by permeant (Takeuchi, 1963) and retain full viability after detergent
cAMP analogues treatment, which kills all amoebae (0-3 % cemulsol for
In mammalian cells, many of the effects of hormones 2h; not shown). Spore formation can be detected at
that use intracellular cAMP as a second messenger can 5mM-Br-cAMP and is half-maximal at llmM-Br-
be mimicked using high extracellular concentrations of cAMP. Of a number of other analogues tested over a
certain cAMP analogues. These analogues can bypass range of concentrations only 8-chlorophenylthio-cAMP
the relevant surface receptor by penetrating the plasma was active. It was roughly as potent as Br-cAMP but
membrane and activating cAMP-dependent protein unfortunately it could not be used above 8 ITIM due to
kinase directly. The most potent analogues, such as precipitation in the incubation medium. The following
those with an 8-substitution of the adenine ring, are were inactive at up to 40 ITIM: C A M P , dibutyryl-cAMP,
effective because they are both more resistant to 8-bromo-cGMP, dibutyryl-cGMP, 2-deoxy cAMP.
hydrolysis by cAMP-phosphodiesterase and better able The experiments described so far show clearly that
to activate the cAMP-dependent protein kinase than Br-cAMP can induce starving cells to differentiate into
cAMP itself (Simon et al. 1973; Miller et al. 1975). The spores, but do not indicate when Br-cAMP (rather than
most promising analogue for Dictyostelium cells seemed cAMP) acts to do this. Three observations suggest that
to be 8-bromo-cAMP (Br-cAMP) which has about a
7-fold increased Km for the phosphodiesterase and a it is the maturation of prespore cells into spores that can
3-fold decreased KA for the protein kinase compared to be specifically promoted by Br-cAMP but not by
cAMP (Van Haastert et al. 1983; de Wit et al. 1982). cAMP. First, cAMP is able to induce starving cells to
Concentrations of 0-1-1 mM-Br-cAMP are usually differentiate as far as prespores but not spores in similar
monolayer incubation conditions (Kay et al. 1978; Kay,
0 10 20 30
8-Br-cAMP concentration (ITIM)
Fig. 1. Induction of spore differentiation
by Br-cAMP in submerged monolayers of
cells of strain V12M2. Left, dose-response
curve with cells at a density of SxlC^cm"2.
Right, phase-contrast micrographs of cells
at lff'cm"2 incubated without Br-cAMP
(top) or with 20mM-Br-cAMP (bottom).
Amoebae were incubated for 48 h in tissue
culture plates containing spore medium
plus 100 ng ml"1 BSA and the appropriate
concentrations of Br-cAMP. Results from 2
dose-response experiments are pooled.756 R. R. Kay
10 r
00
00
a.
Q
D
102 103 104
30 60 90 120 Cell density (cells cm" 2 )
Time (min)
Fig. 3. Cell-density dependence of spore differentiation in
Fig. 2. Induction of UDP-galactose epimerase, a marker strain V12M2. Cells were plated at the stated densities in
for culmination, by Br-cAMP. Migrating slugs at t18 were tissue culture dishes containing spore medium plus 15 ITIM-
partially disaggregated in spore medium and the suspension Br-cAMP, 100 jig ml"1 BSA and 10/igmP1 of the spore
shaken at 180revmin~L with the additions indicated (cyclic germination inhibitor discadenine. Cell differentiation was
nucleotides were 15 ITIM). Duplicate l'5ml portions were scored microscopically at t^. At low cell densities most cells
assayed for enzyme activity and proteins as described in become spores, whereas at high density DIF accumulates
Materials and methods. The experiment is representative of and stalk cells differentiate in consequence. Spores:
4. • • ; stalk cells: A •.
1982). Second, Br-cAMP induces prespore cells, taken tiation might require, in addition to Br-cAMP, some
from migrating slugs, to differentiate into spores with a form of interaction between the cells in the monolayer.
delay of only 3-4 h compared to the 16 h delay with The interaction might require either cell-cell contact or
vegetative cells. Again, spores do not form with cAMP the accumulation of a diffusible inducer but in either
(not shown). Finally a biochemical marker for spore case it would be attenuated at low compared to high cell
maturation, UDP-galactose epimerase (Newell & Suss- density. Fig. 3 shows that spore differentiation in strain
man, 1970), is rapidly induced when Br-cAMP is added V12M2 is in fact very efficient at low densities, where
to slug cells in shaken suspension (Fig. 2). In these the cells are all single. This result is similar to that
conditions cAMP does not induce the enzyme, though obtained previously with various sporogenous mutants
it does stabilize existing levels. (Kay, 1982) and seems to preclude any essential role in
spore induction for cell-cell contact or diffusible
Mode of action of Br-cAMP inducers in these conditions. The reduced efficiency of
Several arguments indicate that Br-cAMP cannot be spore formation at high cell density is probably due to
inducing spore maturation solely by occupation of the accumulated DIF diverting the amoebae to stalk forma-
known surface cAMP receptor: (1) receptor saturating tion (see Fig. 4). Spore formation by cells of strain NC4
concentrations of Br-cAMP (2mM, about 20 times the is always less efficient than with V12M2 cells, being
receptor Ko for Br-cAMP; Van Haastert & Kein, 1983) rarely greater than 30 % at high cell density and falling
do not induce spore maturation (Fig. 1); (2) high to zero at 103 cells cm" 2 (not shown). One contributing
concentrations of agonists (cAMP, 2'-deoxy-cAMP) factor is that the NC4 spores tend to hatch out to give
with a much greater affinity for the surface receptor amoebae soon after they form. Hatching can be
than Br-cAMP are without effect; (3) spore induction reduced by including 10^M-discadenine (a spore germi-
by Br-cAMP is not inhibited by equimolar cAMP, nation inhibitor, Abe et al. 1976) in the medium, but
though this should displace nearly all the Br-cAMP even in this case NC4 cells do not form spores at low
from the surface receptor (the K& for cAMP is about density, suggesting that an additional factor is necessary
450-fold lower than that for Br-cAMP; van Haastert & (see Grabel & Loomis, 1978; Mehdy & Firtel, 1985;
Kein, 1983; result not shown). It therefore seems most Berks & Kay, 1988). The putative factor has not been
likely that Br-cAMP works by penetration of the cell characterized but preliminary experiments indicate that
membrane and activation of the intracellular response it is not methionine or ammonia, which do not improve
machinery in Dictyostelium, as in mammalian cells. the efficiency of spore formation by low-density NC4
cells at 5mM and 20 mM, respectively (not shown;
Involvement of other signals Gibson & Hames, 1988; Gross et al. 1983).
The technique just described for inducing wild-type DIF-1 (l-[3,5-dichloro-2,6-dihydroxy-4-methoxy-
cells to differentiate into spores allows a number of phenyl]hexan-l-one; Morris et al. 1987) is an endogen-
further questions to be asked about the factors control- ous stalk-specific inducer which has been shown to
ling spore differentiation. For instance, spore differen- inhibit prespore and spore differentiation, diverting theInduction of spore maturation by cAMP in Dictyostelium 757
3? 100 drop in ammonia levels can trigger culmination
(Schindler & Sussman, 1977) and would be expected to
produce an elevation in intracellular cAMP levels
(Williams et al. 1984).
Second, the hypothesis suggests a number of lesions
that might account for the sporogenous and rde pheno-
types. Since all the mutants tested are genetically
recessive, they could represent the knock-out of differ-
ent inhibitory elements in the cAMP signal transduction
pathway. Targets might include a G( protein affecting
5 10 15 adenyl cyclase, the regulatory subunit of cAMP-depen-
DIF concentration (nM) dent protein kinase and intracellular cAMP phosphodi-
esterase.
Fig. 4. DIF-1 diverts cells of strain V12M2 from spore to Finally, intracellular cAMP may stimulate differen-
stalk cell differentiation. Vegetative cells were incubated in tiation at other stages of development apart from
tissue culture dishes at a density of SxKh'cm" in spore during culmination (Sampson etal. 1978; Kessin, 1977).
medium supplemented with 100 /.ig ml"1 BSA, 20mM-Br-
cAMP and DIF-1 as indicated and spores scored This idea is attractive even though it has been shown
microscopically after 40 h. The results of 2 experiments, that the expression of certain aggregative and postag-
each done with duplicate plates, are combined. • • gregative genes can be induced without the normal
spore cells; • • stalk cells. DIF-1 also suppressed oscillatory increases in intracellular cAMP (Wurster &
spore formation when slug-stage cells were incubated under Bumann, 1981; Oyama & Blumerg, 1986). Even in
the same induction conditions (not shown). these cases, adenyl cyclase is sufficiently active to
produce intracellular concentrations of cAMP in the fiu
cells to differentiate instead into stalk cells (Kay & range, which should be adequate to stimulate fully the
Jermyn, 1983). It has been suggested that the inhibition cAMP-dependent protein kinase or other cAMP-bind-
of spore differentiation by DIF-1 is a consequence of an ing proteins (Sampson, 1977; de Gunzberg & Veron,
inhibition of cAMP binding to its surface receptor 1982; Tsang & Tasaka, 1986). A role for intracellular
(Wang et al. 1986). Such an inhibition would be cAMP at earlier stages of development is further
bypassed by Br-cAMP if it acts intracellularly. How- suggested by the acceleration of early gene expression
ever, since spore formation induced by Br-cAMP is still in the rde mutants (Sonneborn etal. 1963; Kessin, 1977;
sensitive to inhibition by DIF-1 (Fig. 4), it appears that Abe & Yanagisawa, 1983).
DIF-1 must also have a target further down the signal Spore differentiation by monolayers of wild-type cells
transduction pathway than intracellular cAMP, at least has not been described before (a preliminary report
at the time of spore maturation. appeared in Kay et al. 1988) and this technical advance
allows a number of further questions to be asked about
the control of spore differentiation. One question is
Discussion whether cell-cell contact is necessary for cell differen-
tiation, as has been suggested by indirect experiments
The hypothesis underlying the experiments described (Mehdy etal. 1983; Chisholm etal. 1984). Cell contact is
here is that spore maturation can be triggered by an clearly not necessary in strain V12M2 since isolated
especial elevation in intracellular cAMP levels. This cells at great dilution form spores efficiently in the Br-
hypothesis was first suggested by the altered cAMP cAMP medium (see also Kay & Trevan, 1981; Kay,
metabolism in mutants where spore maturation occurs 1982, for similar results with sporogenous mutants).
more readily than in the wild type and is strongly Strain NC4 differs from V12M2 in that both spore and
supported by the induction of spore maturation in wild- stalk cell differentiation (Berks & Kay, 1988) are very
type strains by permeant cAMP analogues. There are inefficient at low cell density. The reason for this is not
several further consequences of this hypothesis. known but it could be due to a stringent requirement for
First, elevated intracellular cAMP levels may trigger a soluble factor early in development whereas in
spore maturation during normal development as well as V12M2 cells this requirement is more relaxed (Grabel
during monolayer incubation. In support of this, several & Loomis, 1978; Mehdy & Firtel, 1985). A second
studies, including one where the aggregates were indi- question is where is the target for the inhibition of spore
vidually staged, show that cAMP levels increase 2- to cell differentiation by DIF-1 (Kay & Jermyn, 1983). In
3-fold as spores mature during culmination (Brenner, principle, this target could be at any point in the signal
1978; Abe & Yanagisawa, 1983; Merkle et al. 1984). It is transduction pathway leading from extracellular cAMP
possible that in the single exception, where only a small to overt spore differentiation and the cAMP receptor
rise in cAMP levels was detected, the strain A3 used did has been suggested as a potential target (Wang et al.
not develop with sufficient synchrony to produce a 1986). However, the present results suggest an ad-
strong increase in cAMP levels (Brenner, 1978). The ditional target below intracellular cAMP in the path-
rise in cAMP levels during culmination could be way. Finally it has been suggested that cell-cycle phase
brought about by a modulation of the basic cAMP at the time of starvation may determine whether an
signalling system by some other signal. For instance, a individual cell differentiates toward a stalk or a spore758 R. R. Kay
cell (Gomer & Firtel, 1987). In the experiments de- differentiation in Dictyostelium, the prespore vacuole. J.
Ultrastruct. Res. 26, 442-453.
scribed here amoebae can be switched from about 90 % KAY, R. R. (1979). Gene expression in Dictyostelium discoideum:
spore to 90 % stalk cell differentiation merely by adding mutually antagonistic roles of cyclic-AMP and ammonia. J.
DIF-1 to the starvation medium. This, and many other Embryol. exp. Morph. 52, 171-182.
results showing cell-type regulation during develop- KAY, R. R. (1982). cAMP and spore differentiation in
ment (e.g. Raper, 1940), indicate that cell-type differ- Dictyostelium discoideum. Proc. nain. Acad. Sci. U.S.A. 79,
3228-3231.
entiation is regulated by interactions between the cells KAY, R. R. (1987). Cell differentiation in monolayers and the
and is not predetermined by intrinsic differences be- investigation of slime mold morphogens. In Methods in Cell
tween them. Biology 28, 433-448 (ed. J. Spudich). New York: Academic
Press.
I should like to thank Jeff Williams, Mary Berks, David KAY, R. R., GARROD, D. & TILLY, R. (1978). Requirements for cell
Traynor, Ines Carrin, Robert Insall, Jenny Brookman and differentiation in Dictyostelium discoideum. Nature, Lond. 271,
Mark Bretscher for comments on the manuscript. 58-60.
KAY, R. R. & JERMYN, K. A. (1983). A possible morphogen
controlling differentiation in Dictyostelium. Nature, Lond. 303,
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