J. Embryol. exp. Morph. 74, 311-319 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
A prestalk-cell-specific acid phosphatase in
Dictyosteliwn discoideum
By AKIKO A. OOHATA 1
From the Imperial Cancer Research Fund, Mill Hill Laboratories, London
SUMMARY
A simple and reliable method of separating and quantifying acid phosphatase activities in
D. discoideum is described, and one of the electrophoretically distinguishable forms of the
enzyme is shown to be specific for prestalk cells. Accumulation of this product commences
some 2-3 h later in development than the prespore specific enzyme UDP-galactose polysaccharide transferase. This finding is discussed in terms of recent observations on the control of
prestalk cell differentiation in this organism.
INTRODUCTION
During development in D. discoideum starved amoebae aggregate to form
multicellular structures consisting of an anterior region of prestalk cells and a
posterior region of prespore cells. Under normal conditions the anterior cells
give rise to the stalk of the fruiting body eventually formed, while the bulk of the
remainder becomes spores (Raper, 1940). It is well established that the ratio of
numbers of prestalk and prespore cells is largely independent of the total size of
the cell aggregate (Williams, Fisher, Mac Williams & Bonner, 1981), and that
surgically produced fragments of individual aggregates (slugs) can regulate to
reestablish the normal ratio of precursor cell types (Raper, 1940; Sampson,
1976). Both results point to the existence of a mechanism which regulates the
proportion of the two cell types. The nature of this mechanism is being investigated in a number of laboratories (for reviews see Loomis, 1975; Mac Williams
& Bonner, 1979; Gross et al. 1981). For such investigations it is becoming essential to have reliable and quantitative measures of the formation of the two types
of differentiated precursor cells. Such criteria exist for prespore cells in the form
of a prespore (and spore) specific antigen (Takeuchi, 1963) and of an enzyme,
UDP-galactose polysaccharide transferase, involved in the formation of this
antigen (Newell, Ellingson & Sussman, 1969). Using these markers it has been
shown that prespore specific gene products begin to accumulate prior to tip
formation when cells have aggregated to form mounds (Takeuchi, Okamoto,
Tasaka & Takemoto, 1978; Kay, 1979) and that from the earliest time of their
1
Author's present address: Biological Laboratory, Kansai Medical University, Hirakata,
Osaka 573, Japan.
312
A. A. OOHATA
appearance prespore cells are absent from the anterior region of the slug
(Takeuchi et al. 1978).
The situation with regard to criteria for prestalk cell differentiation is much
less satisfactory. Several enzymes have been reported to show higher levels of
activity in prestalk cells than in prespore cells (Krivanek & Krivanek, 1958;
Jefferson & Rutherford, 1976; Armant, Stetler & Rutherford, 1980; Tsang &
Bradbury, 1981). However, these enzymes are not useful as prestalk cell markers
because the differences in activity between the two cell types are not large or the
enzymes are already present in vegetative amoebae. Moreover, although accumulation of prestalk specific polypeptides has been demonstrated by 2-D gel
electrophoresis (Alton & Brenner, 1979; Morrissey, Farnsworth & Loomis,
1981) this method is time consuming and hard to quantify. In a previous publication I identified a unique form of acid phosphatase present in the anterior region
of the slugs of another Dictyosteliwn species, D. mucoroides (Oohata &
Takeuchi, 1977). However in this species mature stalk cells are produced during
migration (Bonner, 1967) so that the slug cells may be more nearly equivalent
to culminating stalk cells than to the prestalk cells of D. discoideum. In the
present work I demonstrate the existence of a prestalk specific form of acid
phosphatase in D. discoideum, and describe a simple, reliable and quantitative
assay for this activity. Evidence is then presented that prestalk cell differentiation, as defined by the accumulation of this enzyme, occurs about 3 h later than
prespore cell differentiation (as evidenced by appearance of the prespore specific
enzyme UDP-galactose polysaccharide transferase).
MATERIALS AND METHODS
Organisms and development
Dictyostelium discoideum strain V12M2 was grown in two-membered culture
with Klebsiella aerogenes on SM agar (Sussman, 1966) at 22 °C for 45 h. Morphogenesis was initiated by plating washed amoebae on 1-8% agar containing
40mM-Na2HPO4-KH2PO4 (pH6-2) and 2-5mM-MgCl2, as described by Tsang
& Bradbury (1981).
Cell separation
Prestalk and prespore cells of the slug were separated by means of a selfgenerating Percoll gradient using the method of Tsang & Bradbury (1981). After
centrifugation, the percentage of prespore cells in each fraction was determined
in order to measure their purity; some of each cell fraction was fixed with
methanol on coverslips and stained with prespore-specific antigen by the method
of Forman & Garrod (1977). The percentage of cells in each fraction that was
stained was determined by fluorescence microscopy.
Prestalk enzyme in D. discoideum
313
Polyacrylamide gel electrophoresis
Cells at various stages of development were harvested in 20mM-KK2
phosphate buffer containing 2mM-MgCl2 (pH6-2), washed with cold distilled
water and stored frozen. For use, cell pellets were thawed and incubated in 0-1 %
of Triton X-100 with occasional vortexing for 20 min at 4 °C, and the lysates spun
in a microcentrifuge for 5 min. Sucrose solution was added to the supernatant to
a final concentration of 15 %.
Polyacrylamide slab gel electrophoresis was performed in essentially the same
way as the Disc electrophoresis described previously (Oohata, 1976). The
following buffer systems were used: 0-2g of imidazole and 5-52 g of barbituric
acid per litre for the running buffer, and imidazole-HCl buffer for the stacking
gel (pH6-2) and the resolving gel (pH7-8). The stacking gel and resolving gel
contained 3-6 % and 6 % acrylamide respectively. The electrophoresis was conducted at 4 °C, and consisted of 15 min at 100 V, followed by 70-90 min at 300 V.
After electrophoresis, acid phosphatases were visualized by the simultaneous
azo-coupling method as follows:- after preincubation in 0-1 M-citric acid-citrate
buffer pH 4-5 for 15 min at room temperature, the gels were incubated for 20 to
30 min at 25 °C in the same buffer at pH 4-8 containing 0-5 mg/ml or 1-5 mg/ml
of a-naphthyl acid phosphate sodium salt (Sigma) and 0-4 mg/ml of fast garnet
GBC (Sigma). The reaction was stopped by replacing the solution with 7%
acetic acid. The stained gels were scanned on a Joyce Loebl Chromoscan 200/
201 system at 520 nm.
Enzyme assays
Glycogen phosphorylase and UDP-galactose polysaccharide transferase were
assayed as described by Tsang & Bradbury (1981) according to the methods of
Town & Gross (1978) and Kay (1979) respectively.
Protein determination
Protein concentration was determined by the method of Bradford (1976).
RESULTS
Identification of electrophoretically distinct acid phosphatases
Extracts of cells of D. discoideum V12M2 at various developmental stages
were prepared and subjected to polyacrylamide gel electrophoresis. Extracts of
vegetative amoebae gave a single band of acid phosphatase (acid phosphatase 1)
which was present throughout development. Another more slowly migrating
band (acid phosphatase 2) was absent in vegetative cells but present in slugs (Fig.
1). Another band which migrated still more slowly than acid phosphatase 2 was
detected in extracts of vegetative amoebae when acetate buffer pH 4-5 was used
EMB74
314
A. A. OOHATA
2 1
Fig. 1. Densitometric tracing of D. discoideum acid phosphatases. Extracts of
V12M2 cells (15/ig protein) were subjected to electrophoresis and the gel stained
histochemically as described in Materials and Methods using l-5mg/ml a-napthyl
acid phosphate as substrate. The arrows indicate the position of the tracking dye. (A)
Vegetative amoebae (to) (B) migrating slugs (tig).
for the enzyme reaction instead of citric acid-citrate buffer (data not shown). It
was not studied further.
The electrophoretic patterns of acid phosphatase in prestalk and prespore cells
To determine whether the developmentally controlled form of acid
phosphatase (acid phosphatase 2) is localized specifically in the prestalk cells of
the slug, as is the case in D. mucoroides (Oohata & Takeuchi, 1977), slug cells
Prestalk enzyme in D. discoideum
315
were disaggregated and separated into prestalk and prespore cells by Percoll
gradient centrifugation. Their electrophoretic patterns were then compared. As
shown in Fig. 2, acid phosphatase 1 was common to both cell types, whereas acid
phosphatase 2 was found only in the prestalk fraction.
Quantitative determination of acid phosphatase activities
The bands obtained by densitometric tracing of stained gels (see Fig. 1) were
found to be symmetrical when up to 30 jug protein extract was loaded. Hence the
absorbance due to acid phosphatase 1 could be estimated from the outside halfarea of its band, and that due to acid phosphatase 2 could be measured in the
same way. Occasionally when band 2 was not well resolved from band 1 its area
was estimated by subtracting twice the half-area of acid phosphatase 1 from the
total area due to the two enzymes. The dependence of reaction rate on protein
A
B
Fig. 2. The electrophoretic pattern of acid phosphatases in prestalk and prespore
cells. The cells of migrating slugs (tn) were dissociated and separated in a Percoll
gradient as described by Tsang & Bradbury (1981). After electrophoresis the gel was
stained for 25 min with 0-5 mg/ml substrate. (A) Whole slugs; in this case all the cells
in one Percoll gradient tube were pooled after centrifugation, and subjected to
electrophoresis. 20ng protein. (B) Top fraction from the gradient; staining with
antispore serum gave 7 % prespore cells. 12fig protein. (C) Bottom fraction; 99 %
prespore cells. 14/^g protein.
316
A. A.
OOHATA
10
iug protein
15
20
Fig. 3. Acid phosphatase 2 activity as a function of protein concentration. Various
dilutions of an extract of migrating slugs (tig) were subjected to electrophoresis and
the enzyme reaction carried out for 30min at 25 °C with 1-5 mg/ml of the substrate
and 0-4mg/ml of fast garnet GBC. The stained gel was scanned at 520 nm and the
absorbance due to acid phosphatase 2 traced onto paper. The absorbance is plotted
in arbitrary units that relate to the area of the tracings of the stained bands.
concentration was determined using this quantitative method. Figure 3 shows a
standard curve of the absorbance due to acid phosphatase 2 as a function of
protein concentration. The reaction was linear to at least 16-5 fig protein with
1 • 5 mg/ml of substrate under the conditions described in Materials and Methods.
A similar result was obtained with acid phosphatase 1.
Kinetics of appearance of the prestalk specific acid phosphatase during development
Figure 4A presents measurements of the activities of the acid phosphatases as
a function of time of development. It can be seen that acid phosphatase 1 activity
dropped to about half its initial value starting at about t 9 , while acid phosphatase
2 remained undetectable until about the same time and then rose to a plateau at
tl8-
In order to relate the time of appearance of the prestalk specific acid
phosphatase 2 to other developmental events the accumulation of two wellknown postaggregative enzymes (Town & Gross, 1978) was examined in the
same experiment. Glycogen phosphorylase, present in both prestalk and prespore cells (Tsang & Bradbury, 1981), and UDP-galactose polysaccharide transferase, a prespore-specific product (Newell et al. 1969), both began to appear
before 19. Thus the appearance of acid phosphatase 2 is delayed by 2-3 h compared to these two enzymes. Similar results to those shown in Figs 4A and 4B
were obtained with strain NC4 of D. discoideum, though the activity of acid
phosphatase 2 was lower than in strain V12M2 (data not shown).
Prestalk enzyme in D. discoideum
TO
317
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^
is o
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30
13 E
"Si
20
6
o—
o
O W
a.-o
O
B
•a wE
i
10
o
D
12
15
18
21
Fig. 4. Changes in specific activity of acid phosphatases during development. (A)
Each value represents the mean of two experiments; open circles, acid phosphatase
1; closed circles, acid phosphatase 2. (B) Open circles, glycogen phosphorylase;
closed circles, UDP-galactose polysaccharide transferase. The abscissa indicates the
times after initiation of development.
DISCUSSION
Multiple forms of acid phosphatase have been identified previously by
Solomon, Johnson & Gregg (1964) and Parish (1976). In the present study, I
have focused attention on the two forms of enzyme detected when the assay is
performed in citrate buffer, and have shown that one of these forms is specific
318
A. A. OOHATA
for prestalk cells. I have not attempted to identify the molecular basis of the
difference between the two enzyme forms. The function of the prestalk-specific
enzyme is unknown, though it may play a role in cell wall formation at the time
of stalk construction (see Gezelius, 1972).
In studies in this laboratory on cell differentiation in monolayers it has been
shown that amoebae incubated at low density in the presence of cyclic AMP
develop into stalk cells only if provided with a low-molecular-weight lipid-like
factor (DIF) that is liberated into the extracellular medium during development
(Town, Gross & Kay, 1976; Town & Stanford, 1979). It has been suggested by
Gross et al. (1981) that DIF is an activator of prestalk cell formation in a lateral
inhibition mechanism of the kind proposed by Gierer & Meinhardt (1972).
Recently, Brookman, Town, Jermyn & Kay (1982) have shown that DIF
accumulates in cells co-ordinately with glycogen phosphorylase and UDPgalactose polysaccharide transferase (a prespore-specific product). My observation that the appearance of acid phosphatase is delayed by two to three hours
compared to these products is thus quite compatible with the idea that prestalk
cell differentiation is dependent upon prior accumulation of DIF. Further
evidence that synthesis of acid phosphatase 2 is DIF-dependent comes from our
recent finding that mutants that fail to make their own DIF, but can respond to
it, also fail to accumulate acid phosphatase 2. Moreover, acid phosphatase 2 is
produced when both DIF and cAMP are added in normal development. Such
mutants do accumulate high levels of prespore specific UDP-galactose
polysaccharide transferase (Kopachik, Oohata, Dhokia, Brookman & Kay (in
preparation)).
I am greatly indebted to Adrian Tsang for advice and technical help. I should like to thank
Babu Dhokia for technical assistance, Mike Peacey for drawing the diagrams, and my other
colleagues at ICRF for helpful discussion. Sincere thanks are also due to Julian Gross for his
valuable discussion and for help in the preparation of the manuscript.
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(Accepted 6 November 1982)