/. embryol. exp. Morph. 78, 229-248 (1983)
229
Printed in Great Britain © The Company of Biologists Limited 1983
The effects of inhibition of protein glycosylation on
the aggregation of Dictyostelium discoideum
By CHARLES JOHN McDONALD 1 AND JEFFREY SAMPSON 2
From the Department of Biochemistry, University of Leicester
SUMMARY
At concentrations greater than 10 fig ml"1 tunicamycin inhibited the incorporation of
[3H]mannose into glycoproteins during the early phase of development in Dictyostelium
discoideum, however, total protein synthesis was unaffected. Tunicamycin also interfered
with the normal process of aggregation. In its presence small aggregates were observed at the
time of normal aggregation, but amoebae failed to aggregate completely and subsequent
development was inhibited. Inhibition of normal aggregation by tunicamycin was found to be
reversible. The appearance of cell-associated and secreted cyclic AMP phosphodiesterase and
cell-surface contact sites A was prevented by tunicamycin but cell surface cyclic AMP receptor
activity developed normally in its presence. Tunicamycin also prevented amoebae from
acquiring the ability to chemotact toward cyclic AMP. Addition of exogenous cyclic AMP
phosphodiesterase restored the ability of amoebae to chemotact toward cyclic AMP in the
presence of tunicamycin. Our data suggest that the primary block in aggregation caused by
tunicamycin results from the inhibition of expression of active cyclic AMP phosphodiesterase.
ABBREVIATIONS
cyclic AMP
dimethyl P O P O P
DMSO
i.u.
NP 40
PPO
SDS-PAGE
TCA
TM
tx
adenosine 3' 5' cyclic monophosphate
l,4-bis-['2-(4-Methyl-5-phenyloxazolyl)-3]-Benzene
Dimethylsulphoxide
International Unit of Enzyme Activity
The non-ionic detergent Nonidet P40
2,5-diphenlyoxazole
sodium dodecyl sulphate polyacrylamide gel
electrophoresis
trichloroacetic acid
Tunicamycin
time in hours (x) after the onset of development
Enzymes
PDE
1
quoted
cyclic A M P phosphodiesterase [E. C. 3.1.4.17]
Author's address: Department of Biochemistry, University of Leeds, Leeds, U.K.
Author's address: Department of Biochemistry, School of Biological Sciences, University
of Leicester, Leicester LEI 7RH, U.K.
2
230
C. J. MCDONALD AND J. SAMPSON
INTRODUCTION
The molecular basis of cellular communication in embryonic development in
higher eukaryotes has been difficult to study because of the close packing and
adhesion of the cells comprising the tissue. Aggregation of the cellular slime
mould Dictyostelium discoideum, however, starts from a field of apparently
identical amoebae (Bonner, 1944) and cell movements continue until the
amoebae are associated into multicellular aggregates (Bonner, 1949 and 1963).
It therefore offers an attractive system for the study of intercellular communication. The molecular basis of aggregation is now well understood (Loomis, 1975;
Robertson & Grutsch, 1981) and the possibility has been raised that this system
may be responsible for cellular communication and movement during the later
multicellular stages of the organism's development (Newell, 1978). Indeed, it
has been a subject of conjecture that the mode of D. discoideum aggregation may
be found among the as yet uncharacterized cellular communication systems of
higher eukaryotes (Robertson & Grutsch, 1981; Gingle, 1977; Guillermet &
Mandaron, 1980).
Aggregation of D. discoideum is initiated by certain amoebae secreting pulses
of cyclic AMP and the surrounding amoebae responding by positive chemotaxis
and the secretion themselves of cyclic AMP pulses (Konijn, 1972). This signal
relay system entrains amoebae over a large area into each aggregation centre into
which cells move in synchronous waves or spirals (Gerisch, 1968; Alcantara &
Monk, 1974; Newell, 1978). The enzymes which control the cyclic AMP signal
are developmentally regulated. Prior to aggregation, adenyl cyclase activity is
present (Klein & Darmon, 1977; Darmon & Klein, 1978) and cyclic AMP
phosphodiesterase and its inhibitor protein are secreted by the amoebae (Pannbacker & Bravard, 1972; Gerisch et al. 1972). Parallel with the acquisition of the
ability to chemotact toward cyclic AMP pulses at the onset of aggregation there
is a rapid increase in cell surface cyclic AMP phosphodiesterase (Malchow et al.
1972). As aggregation proceeds the synchronous moving waves of amoebae
break up into 'streams' (Bonner, 1971), stream formation being mediated
through end-to-end cell contact via cell surface contact sites A which also increase in activity during this period (Beug et al. 1973). There is evidence that the
cell surface aggregation functions (cyclic AMP receptors, cyclic AMP phosphodiesterase and contact sites A) are synthesized de novo during aggregation
(Sampson et al. 1978) as are a large number of secreted enzymes which increase
in activity during early development (Loomis, 1975).
Cell surface cyclic AMP phosphodiesterase (Eittle & Gerisch, 1977), the
secreted form of the enzyme (Orlow et al. 1981), its inhibitor (Franke & Kessin,
1981) and contact sites A (Muller & Gerisch, 1978) are all glycoproteins as are
many membrane components which change during development (Toda et al.
1980). In order to study the role of de novo protein glycosylation during aggregation in D. discoideum we have used the antibiotic tunicamycin which inhibits the
Tunicamycin and the development of Dictyostelium
231
first step in the glycosylation of proteins via N-asparaginyl links (Takatsuki et al.
1971; Tkacz & Lampen, 1975). We report that inhibition of protein glycosylation
by tunicamycin (TM) prevented the appearance of cyclic AMP phosphodiesterase and contact sites A but that cell surface cyclic AMP receptor activity
developed normally. TM caused a reversible block in development but small
aggregates did form in the presence of TM at the normal time of aggregation. TM
prevented the development of the ability of amoebae to chemotact toward cyclic
AMP but the addition of cyclic AMP phosphodiesterase to TM-inhibited cells
restored this activity. The implications of these results are discussed.
MATERIALS AND METHODS
Chemicals
Tunicamycin was the kind gift of Drs Harper and Herrman, Biotechnology
Department, Glaxo Research Limited, Greenford, Middlesex, England. It was
stored either desiccated as a powder or as a 1-5 jug ml" 1 solution in DMSO at
—20 °C. DMSO at concentrations up to 0-44 M had no observed effect on
development of Dictyostelium or on any of the cellular or biochemical functions
measured in this study. Radiochemicals were obtained from Radiochemicals
International, Amersham, England. All other chemicals and reagents were of
the highest purity available.
Growth and development of the organism
Dictyostelium discoideum strain AX2 spores (a kind gift from Dr M. North)
were stored on silica gel at 4°C. Germination and growth were carried out in
aerated suspensions of HL5 growth medium (1 %(w/v) peptone, 0-5 % (w/v)
yeast extract, 20 mM-phosphate buffer, pH 6-9) containing 86 mM-glucose (Watts
& Ash worth, 1970). The media contained 200 /j.g ml" 1 streptomycin and 5 fj.g ml" 1
tetracycline. Flasks, at least four times the volume of suspension, were shaken
at 175 r.p.m. on a New Brunswick G2 gyrotary shaker. Amoebae were harvested
during log phase (2-4 x 106 cells ml"1) by centrifugation at 800 g for lOmin at
22°C, washed three times in KK2 buffer (20mM-potassium phosphate, pH6-2)
containing 0-5 mM-CaCb and 2 mM-MgSO4, and finally resuspended in this same
buffer at a concentration of 1 x 107ml~1. Washed amoebae were shaken at
125 r.p.m. and 22°C in chromic acid washed, siliconized flasks and the onset of
development (t0) was measured from the time of final resuspension of the washed
amoebae. To observe aggregation, washed amoebae were plated onto 1-2%
agarose in KK2 buffer at a density of 5 x 105 amoebae cm" 1 .55 mm tissue culture
dishes were used, each containing 3 ml agarose. Photomicrography was performed using a Wild dissecting microscope fitted with a Nikon cameraback.
Measurement of chemotaxis
The small population test was used (Konijn etal. 1969). 1 //I drops of amoebae
232
C. J. MCDONALD AND J. SAMPSON
4
(2 x 10 cells) were placed on the surface of 0-5 % agarose in KK2 buffer at a
distance of 0-5-1-5 mm from an equal volume of cyclic AMP of the stated concentration. Plates were incubated at 22 °C for 3h. The rate of chemotaxis was
then measured in mmh" 1 using an ocular micrometer.
Protein synthesis and protein glycosylation
3
5/xCi L(4,5- H)leucine (40-60 Ci-mmole1) and 2^Ci D(U-14C)mannose
(200-300 mCi mole"1) were added to 10 ml of developing amoebae at t2.
Tunicamycin in DMSO, DMSO alone or KK2 was added at t3 then duplicate
0-5 ml samples were removed at U and thereafter as stated. Nonidet P40 was
added to each sample to a final concentration of 0-2%, samples were mixed
vigorously for 5 sec and then an equal volume of 10 % TCA containing 1 mg ml" 1
each of leucine and mannose was added. TCA precipitates were collected onto
Whatman GF/C filters and washed in 5 % TCA containing lmgml" 1 each of
leucine and mannose. The filters were dried and counted in PPT scintillant
(15gPPO, 18-8mg dimethyl POPOP per 2-5 litre toluene). The relative rates of
incorporation of [14C]mannose and [3H]leucine into TCA precipitates were
determined by double isotope analysis (Neame & Home wood, 1974) and were
used respectively as a measure of protein glycosylation and protein synthesis.
Measurements of aggregation functions
Total cell adhesion was measured by resuspending amoebae at a concentration of 106 cells per ml in KK2 buffer. Cell clumps were disaggregated by three
passages through a 25G x 5/8' needle. Total cell number was determined by
haemocytometer counting. The cell suspension was then shaken at 250r.p.m.
for 30 min on a New Brunswick gyrotary shaker (model G2, 3/4' circular
orbit). The number of single cells remaining after this period was then
determined. Total cell adhesion was then calculated by subtracting the number
of single cells left after 30 min from the total number of cells and expressing
this figure as a percent of total cells. Contact sites A were measured by
performing the above assay in KK2 lacking divalent cations and in the presence
of 10mM-EDTA (Rosen et al. 1973). A subtraction of the adhesion due to
contact sites A from total cell adhesion provided an estimate of adhesion due
to contact sites B (Marin et al. 1980). This estimate is only valid at low contact
sites A activity. Cyclic AMP phosphodiesterase was assayed as described
(Henderson, 1975). The cell-associated enzyme activity was taken as a measure
of the enzyme on the cell surface (Malchow et al. 1972) and was expressed as
milli International units (/imoles cyclic AMP hydrolysed per minute per mg
protein at 37 °C). The activity of the extracellular enzyme was expressed as
//moles cyclic AMP hydrolysed per minute per ml of extracellular fluid at 37 °C.
Cell surface cyclic AMP receptors were assayed as described (Green & Newell,
1975).
Tunicamycin and the development of Dictyostelium
233
Protein analysis and the preparation of cyclic AMP phosphodiesterase
Suspensions of developing amoebae were perfused with a solution of cyclic
AMP which was delivered by an LKB microperspex pump (model 2132) at the
rate of 50 [A per minute such that the concentration of cyclic AMP following the
first drop was 0-2 fjM. This treatment maximizes the secretion of cyclic AMP
phosphodiesterase and represses the production of the protein inhibitor
(Gerisch, 1979). After fifteen hours of development the suspension was cleared
of cells by centrifugation at 800 g for lOmin at 22 °C. The cell pellet was resuspended in 50 mM-tris-HCl, pH 7-5 (T7.5 Buffer) and the cells lysed by the addition
of NP 40 to a final concentration of 0-2 % (v/v). This lysate served as the source
of cellular proteins. The extracellular fluid was cleared of debris by centrifugation at 10000g for lOmin at 4°C. Protein was precipitated by the addition of
ammonium sulphate to 90 % saturation. The precipitate was collected, resuspended in T7.5 buffer and dialysed against 200 volumes of the same buffer. This
solution provided the source for the analysis of extracellular proteins and, after
passage through a column of concanavalin A Sepharose as previously described
(Kessin et al. 1979), provided a partially purified extract of cyclic AMP
phosphodiesterase, 30i.u. of cyclic AMP phosphodisterase were obtained from
100 ml of an amoebal suspension.
Protein determinations were carried out by the method of Lowry (Lowry etal.
1951) and the secreted polypeptides were analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (Laemmli, 1970) followed by silver
staining of the separated proteins (Oakley et al. 1980).
RESULTS
The effect of tunicamycin on protein synthesis and protein glycosylation
We studied the effect of tunicamycin (TM) on the incorporation of
14
[ C]mannose and pH]leucine into TCA-precipitable material in order to
monitor its effects on both protein synthesis and glycosylation. Mannose was
chosen because it is the major sugar constituent of N-linked oligosaccharides, the
synthesis of which is inhibited by TM. [14C]mannose and [3H]leucine were added
to cell suspensions 2h after the onset of development (t2). TM was then added
at t3 to final concentrations ranging from l^gm!" 1 to 40iugml~1. The amounts
of radioisotopes incorporated were then determined. The results (Table 1) show
that TM up to a concentration of 40/igml" 1 had no significant effect on the
incorporation of pHjleucine into TCA-precipitable material. However, increasing concentrations of the antibiotic markedly inhibited the incorporation of
[14C]mannose. A concentration of 5/igml" 1 inhibited the incorporation of
[14C]mannose by 50 %, near maximal inhibition being obtained at concentrations
of 10/igml" 1 or greater. We take these data to show that TM inhibits protein
234
C. J. MCDONALD AND J. SAMPSON
Table 1. The effect of tunicamycin concentration on the rate of incorporation of
[3H]leucine and [14C]mannose into TCA-precipitable material by amoebae from
t4 to t9 of development
Tunicamycin
concentration
(^gml-1)
Rate of incorporation
of [3H]leucine
(% control)
Rate of incorporation
of [14C]mannose
(% control)
0
1
2-5
5
10
20
40
100
136
92
124
98
124
88
100
83
53
49
5
12
13
The conditions of labelling and analysis were as outlined in the Materials and Methods. The
control rates for [3H]leucine and [14C]mannose incorporation were 135 d.p.m./107 cells/h and
94d.p.m./107 cells/h, respectively.
glycosylation in Dictyostelium but has little or no effect on total protein
synthesis. Thus, it appears that TM affects D. discoideum in exactly the same
way that it effects other eukaryotic cells. The effect of TM on the time course of
protein synthesis and glycosylation is shown in Fig. 1. There was no appreciable
effect of TM on the incorporation of [3H]leucine (Fig. 1A), but, under the
conditions of the assay, TM reduced the rate of [14C]mannose incorporation to
4 % of the control rate. This low level incorporation of [14C]mannose into TCAprecipitable material in the presence of TM may reflect the general metabolism
of mannose or the incorporation of the sugar into ohgosaccharide chains that are
attached to serine or threonine residues in proteins via O-glycosidic bonds. Such
incorporation would be expected to be TM insensitive. Treatment of TCA
precipitates with 25 % 2-methoxyethanol (v/v) to solubilize large molecular
weight carbohydrate did not reduce this low level, TM insensitive [14C]mannose
incorporation (data not shown).
The effects of TM on development
When TM (10-40 jug ml"1) was added to suspensions of developing amoebae
between to and U, and the cells then plated on agarose containing an equivalent
concentration of the antibiotic, aggregation was severely impaired and all
subsequent development was blocked. Amoebae failed to aggregate fully, instead, small aggregates began to form at about the same time as control amoebae
aggregated (Fig. 2). The formation of these small aggregates was very rapid,
proceeding from initiation to completion in 30 to 40min (Fig. 3). These small
aggregates often resembled an interconnected array of flat areas of amoebae
(e.g. Fig. 2B and Fig. 4A) but further development did not occur. However, if
Tunicamycin and the development of Dictyostelium
235
A.
400
300
« E 200
.S d
I?
100
L
T
2
250
4
6
8
1
B.
200
150
/
/
100 -
• /
u
50
L
2
T
#
/
/
4
6
8
Hours of development
10
Fig. 1. The effect of tunicamycin on the incorporation of pHjleucine and
[14C]mannose by developing amoebae. Labelling conditions were as described in the
Materials and Methods section. (A) [3H]leucine incorporation; (B) (14C]mannose
incorporation into TCA precipitable material. 'L' denotes the time of addition of the
labelled compound and T ' denotes the time of addition of tunicamycin to a final
concentration of lOjUgml"1. ( •
• ) control amoebae; ( • — • ) tunicamycintreated.
amoebae were exposed in suspension to TM from to to tio then plated as
drops on agarose lacking the antibiotic, normal-looking fruiting bodies were
observed 48 h later, showing that TM does not cause an irreversible block in
development. TM also affected aggregation and subsequent development at
concentrations below that required for complete inhibition of protein glycosylation (i.e. lOjugml"1; Table 1). The small, abnormal aggregates were observed
in the presence of TM at concentrations ranging from ^ n g m l " 1 to 40 jugml"1.
Their formation began at the same time as aggregation of control amoebae and
was always complete within 60min, although cell movements were still observed after this time and the small aggregates very often fused together into an
236
C. J. MCDONALD AND J. SAMPSON
'«U&
tmm*mmmm**~ *
Fig. 2. The effect of tunicamycin on aggregation. Aggregation was allowed to
proceed on agar plates and the photographs were taken at tio. The horizontal bar
represents a distance of 2mm. (A) Control amoebae, (B) small aggregates formed
1
in the presence of tunicamycin
interconnected array. This process was usually complete by ts and was most
marked at TM concentrations below 5/igml" 1 . No further development was
observed within the flat arrays at TM concentrations above l^SjUgml"1 but at
lower antibiotic concentrations mounds of cells were observed at t22 which were
larger and more numerous at the lowest TM concentrations studied (Figs 4A, B
and C). These mounds had developed between ts and t22 by apparently drawing
in amoebae from the surrounding flat arrays but they did not develop further. At
the lowest TM concentrations (40 ng ml" 1 to 0-16 /jg ml"1) nearly all the amoebae
present initially in the flat arrays had collected into mounds by tn and 'finger-like'
projections could be seen protruding from these mounds (Fig. 4D). These structures failed to develop further. Thus it appears that TM blocked development at
all the concentrations studied but that the precise point of the block depended
on the concentration of TM.
The effect of TM on the development of aggregation functions
If TM (final concentration of 10/igml"1) was aded to amoebae developing
in suspension they subsequently failed to acquire cell-associated cyclic AMP
phosphodisterase and contact sites A activity (Fig 5A and 5B, respectively) but
they did acquire near normal levels of cell surface cyclic AMP receptors (Fig.
5C) Amoebae developing on agarose containing TM also failed to accumulate
Tunicamycin and the development of Dictyostelium
237
Fig. 3. Time course of appearance of the small aggregates in the presence of
tunicamycin (10 /ig ml"1). Amoebae were allowed to develop on agar in the presence
of 10/igml"1 tunicamycin. The horizontal bar represents a distance of 40jum. (A)
Single cells observed at ts-75, (B) aggregation beginning to take place at ts-9, (C)
definite aggregates observed at t6 , (D) typical final appearance of small aggregates
viewed at U-s •
238
C. J. MCDONALD AND J. SAMPSON
Fig. 4. The effect of different concentrations of tunicamycin on the development of
amoebae on an agar surface. Amoebae were allowed to develop on agar containing
different concentrations of tunicamycin and photographs were taken after 22 h
development. Arrows indicate the mounds that occur within the flat arrays of cells.
In A, B and C the magnification used was x6 and the horizontal bar represents 2 mm;
in D the magnification used was x50 and the bar represents 40 /im. A. 5 jug ml"1; B.
0-6/igmr 1 ; C. 0-3/igmr 1 ; D. 40ngml~1.
Tunicamycin and the development of Dictyostelium
239
cell-associated cyclic AMP phosphodiesterase and contact sites A (data not
shown). Addition of TM to amoebae during the normal developmental increase
in cyclic AMP phosphodiesterase and contact sites A activity prevented any
further accumulation of these two activities (data not shown), indicating that the
antibiotic has an almost immediate inhibitory effect on their expression.
We consistently observed a slight decrease in total cell adhesion during the first
three hours of development which then increased as contact sites A (csA) activity
increased (Fig. 6). Since the level of csA is very low during the first three hours,
this decrease must be due to a slight reduction in contact site B (csB) mediated
adhesion. Our estimate of csB activity is only valid at low csA activity therefore
we are unable to plot csB activity at later stages of development, however, csB
activity has been reported to remain fairly constant throughout development
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Fig. 5. The effect of tunicamycin on the acquisition of aggregative functions. The
arrows mark the time of addition of tunicamycin to a final concentration o(
lOjUgml"1. All enzyme activities were assayed as outlined in the Materials and
Methods section. (A) Cell-surface cyclic AMP phosphodiesterase, (B) contact sites
A, (C) cell-surface cyclic AMP receptors. ( •
• ) control amoebae; ( •
•)
tunicamycin-treated.
240
C. J. MCDONALD AND J. SAMPSON
100 r A. CONTROL
80
60
40
20
2
100 r
4
6
8
10
B. TUNICAMYCIN-TREATED
80
60
40
20
10
4
6
8
Hours of development
Fig. 6. The effect of tunicamycin on cell cohesion during development. Tunicamycin
was added at the beginning of development (to) to afinalconcentration of 10 jug ml"1.
(A) Control amoebae; (B) tunicamycin-treated. (O
O) Total cohesion,
(•
• ) EDTA-sensitive cohesion (contact sites B), (A
A) EDTA-resistant
cohesion (contact sites A).
2
Fig. 7. The effect of tunicamycin and exogenous cyclic AMP phosphodiesterase
(PDE) on the chemotaxis of amoebae toward cyclic AMP. Chemotaxis tests were
performed as outlined in Materials and Methods section. The bar represents a distance of 2 mm. The centre of the original amoebal droplet is marked by the asterisk
and the boundary of the original droplet is marked by the broken line. Tunicamycin
was added at a concentration of 10 jugml"1 where appropriate. Arrows indicate the
front of the responding amoebae, the cyclic AMP source being on the right of the
picture in each case. (A) Chemotaxis of control amoebae toward 0-05 mM-cyclic
AMP; (B) chemotaxis of control amoebae toward 5 mM-cyclic AMP in the presence
of 3-6 milli i.u. of exogenous PDE; (C) the response of tunicamycin-treated amoebae
toward 0-25 mM-cyclic AMP; (D) chemotaxis of tunicamycin-treated amoebae
toward 5 mM-cyclic AMP in the presence of 3-6 milli i.u. of exogenous PDE.
Tunicamycin and the development of Dictyostelium
1
241
(Beug et al. 1973). Amoebae incubated in the presence of lO/igml" TM from
to lost all cohesion by t3 (Fig. 6B), the rate of loss being more rapid in the
presence of TM than the early loss of adhesion of control amoebae (compare Fig.
6A and 6B). Thus de novo protein glycosylation would appear to be necessary
Fig. 7
242
C. J. MCDONALD AND J. SAMPSON
not only for the developmental appearance of csA but also for the maintenance
of csB activity throughout development.
The effect of TM on amoebal chemotaxis toward cyclic AMP
Figure 7 shows the effect of TM on the ability of amoebae to chemotact toward
cyclic AMP as measured by the 'small population test' (Konijn et al. 1969).
Amoebae were allowed to develop from to in suspension in either the presence
or absence of TM (lO/zgml"1). At t4 amoebae were removed from suspension
and deposited on chemotaxis plates as described in the "Materials and Methods"
section. Plates were examined 3h later for signs of chemotaxis. All chemotaxis
tests for TM-treated amoebae were performed on agar containing 10/igml" 1
TM. Control amoebae responded to 50 /iM cyclic AMP by positive chemotaxis at
a rate of 0-73 mm rT 1 over the 3 h period (Fig. 7A). TM-treated amoebae failed
to chemotact significantly toward any cyclic AMP concentration in the range
Table 2. The rates of chemotaxis of amoebae toward different concentrations of
cyclic AMP in the presence and absence of tunicamycin (10 pig ml'1) and
exogenous cyclic AMP phosphodiesterase (PDE)
A. Chemotaxis in the absence of cyclic AMP phosphodiesterase
Cyclic AMP
concentration
Chemotaxis (mm h l)
—K~
\
Control amoebae
Tunicamycin-treated amoebae
(ITIM)
0
0
0-05
0-10
0-25
0-50
0-75
0-73
0-47
0-42
0-42
0-11
0.
0.
**
**
**
0.
B. Chemotaxis inthe presence of cyclic AMP phosphodiesterase
Chemotaxis (mmh" 1 )
Cyclic AMP
concentration
K
c
Control amoebae
\
Tunicamycin-treated amoebae
(HIM)
5
10
25
50
0-3
0-42
0-48
0
1-3
1-7
1-4
1-8
Chemotaxis tests were performed as outlined in the Materials and Methods section. The
concentrations of cyclic AMP given are those which elicited an amoebal response under the
various conditions used.
** Denotes a response of TM-treated amoebae to cyclic AMP, as discussed in the text,
without measurable chemotaxis.
Tunicamycin and the development of Dictyostelium
68—
243
—p65
40_
22—
A
B
Fig. 8. Analysis of total secreted proteins from cells developing in the presence or
absence of tunicamycin (lO[igm\~l). Total secreted proteins were prepared and
analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis as outlined
in the Materials and Methods section. Proteins were detected using the ultrasensitive
silver stain method (Oakley et al. 1980). The position of migration of the different
marker proteins and their relative molecular masses are given on the left-hand side
of the figure and the position of the major polypeptides whose secretion is inhibited
by tunicamycin (p65, p52 and p50) is indicated on the right-hand side. 5 jug of protein
was applied to each track. (A) In the presence of tunicamycin (10 fig ml"1), (B) in the
absence of tunicamycin.
244
C. J. MCDONALD AND J. SAMPSON
50/iM to 50 mM. However, amoebae were consistently observed to respond to
cyclic AMP in the range 0-1 mM to 0-5 mM by moving out slightly from the
original confines of the droplet toward the cyclic AMP source (Fig. 7C).
The effect of exogenous cyclic AMP phosphodisterase (PDE) on chemotaxis
was also studied. Approximately 4 milli i.u. of partially purified extracellular
PDE was added to the amoebal droplet used in the chemotactic tests. In the
presence of PDE, the threshold cyclic AMP concentration below which control
amoebae would not chemotact was raised approximately 100-fold (Fig. 7B).
Addition of the enzyme to TM-treated cells resulted in amoebal chemotaxis
toward cyclic AMP concentrations ranging from 0-5 mM to 50 mM (Fig. 7D). The
chemotactic rates of control and TM-treated amoebae toward a range of cyclic
AMP concentrations under these different conditions are summarized in Table
2. Comparison of Figs 7C and 7D also illustrates that in the presence of PDE TMtreated cells that are left within the confines of the original droplet formed into
very compact, small aggregates instead of the usual rather amorphous field of
small aggregates. This same difference in morphology was observed when TMtreated cells were placed on agar in the absence of a droplet of cyclic AMP (data
not shown).
Polypeptides secreted by TM-treated amoebae
Amoebae were allowed to develop in suspension for 15 h either in the presence
or absence of tunicamycin. Developing cells were perfused with cyclic AMP
throughout the 15 h period in order to maximize the production of extracellular
cyclic AMP phosphodiesterase (see Materials and Methods). At tis cellular and
secreted proteins were processed for analysis. TM-treated cells contained 10 %
more protein than controls but secreted 50 % less protein than controls (data not
shown). We were unable to detect any extracellular cyclic AMP phosphodiesterase activity in TM-treated cells, even after the removal of any
unhydrolysed cyclic AMP from the extracellular fluid. Analysis of secreted
proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed
that TM-treated cells failed to secrete three major polypeptides under the conditions used (Fig. 8). These polypeptides had apparent relative molecular masses
of 65 000 (p65), 52 000 (p52) and 50 000 (p50). No polypeptides were observed
in the secreted extracts of TM-treated amoebae which were not also present in
the secreted extracts of controls. There is evidence to show that p52 and p50 are
closely related glycoprotein subunits of the extracellular cyclic AMP phosphodiesterase (C. J. McDonald and J. Sampson, manuscript in preparation).
DISCUSSION
In this paper we report that during the aggregation phase of development in
Dictyostelium discoideum tunicamycin (TM) at concentrations above lO/igmP 1
inhibited protein glycosylation but had little or no effect on protein synthesis.
Tunicamycin and the development of Dictyostelium
245
This inhibition of protein glycosylation was accompanied by a reversible block
in development. These data substantiate a preliminary report that TM blocked
the development of D. discoideum (Toda et al. 1980). Two recent studies have
shown that TM inhibited protein glycosylation in D. discoideum although as
concentrations of only 3 fig ml"1 (Lam & Siu, 1982) and 5 //gml" 1 (Yamada et al.
1982) were used, complete inhibition was not achieved. In all other eukaryotes
studied so far, TM specifically inhibited the first step of the N-asparaginyl-linked
pathway of protein glycosylation (Lennarz, 1980). It is therefore reasonable to
assume that TM inhibition of protein glycosylation in D. discoideum occurs via
a similar mechanism.
The effect of TM on aggregation was very much concentration dependent. At
10/igml" 1 , which caused maximal inhibition of protein glycosylation, aggregation was severely impaired and only very small aggregates developed; all further
development was blocked. At TM concentrations below lO^gml" 1 protein
glycosylation was incompletely inhibited and the small aggregates of cells were
observed to collect together into larger interconnected arrays of cells. At TM
concentrations below 1-25 fig ml" 1 mounds of cells developed within these interconnected arrays and at the lowest antibiotic concentrations tested ^ O n g m r 1 )
multiple 'finger-like' projections developed from these mounds by t22, but
development did not progress further. The morphology of aggregation of
amoebae from the flat arrays into mounds and 'finger-like' structures at subinhibitory concentrations of TM may be grossly aberrant, but the phenomenon
could represent a slower version of normal aggregation caused by the slower
accumulation of glycoproteins necessary for the process. Defects in the process
would arise due to glycoproteins not reaching the required concentration for the
formation of normal structures and normal levels of enzyme activity. We suggest
that the small aggregates which form regardless of the concentration of TM may
represent a transitional stage in aggregation which is blocked from further
development by the absence of specific glycoproteins which are crucial for normal development.
TM prevented the acquisition of two glycoproteins implicated in normal
development:- cyclic AMP phosphodiesterase and contact sites A. Indeed, the
small aggregates formed in the presence of TM were reminiscent of those formed
by aggregation-deficient mutants which lack cyclic AMP phosphodiesterase
(Riedel et al. 1973). Cyclic AMP phosphodiesterase activity has been shown to
be required for normal aggregation: an aggregateless mutant deficient in the
enzyme, HPX235, can aggregate when supplied with an exogenous source of
cyclic AMP phosphodiesterase (Darmon et al. 1978), and aggregation can be
blocked by a specific neutralizing anti-cyclic AMP phosphodiesterase antibody
(Goidl et al. 1972). Amoebae acquired cyclic AMP receptors normally in the
presence of TM and when PDE was added to them they regained the ability to
chemotact toward cyclic AMP, formed more compact aggregates but did not
develop further. We therefore suggest that the primary block in aggregation
246
C. J. MCDONALD AND J. SAMPSON
induced by TM treatment is to prevent the synthesis of active PDE and that the
subsequent block in development is perhaps caused by either a defect in regulation of the enzyme's activity or by the lack of cell cohesion due to the inhibition
of the appearance of contact sites A. The formation of small aggregates which
we observed on agarose containing TM may be explained by the diffusion of
secreted cyclic AMP into the agarose thus preventing the immediate saturation
of cyclic AMP receptors and allowing amoebae to respond to a limited number
of chemotactic signals. Similar small aggregates were not observed when
amoebae were spread on nitrocellulose filters (Lam & Siu, 1982) although it is
interesting that in that study amoebae were observed to 'orientate' pseudopodia
towards cyclic AMP in chemotactic tests.
The normal acquisition of cyclic AMP receptor activity in the presence of TM
implies that de novo protein glycosylation is not necessary for the proper expression of its function. This does not rule out a regulatory role for preexisting
glycoproteins such as contact sites B (Marin et al. 1980) but as cell cohesion
rapidly decreased during early development in the presence of TM, if such a role
existed, one might expect at least a slower accumulation of receptor activity and
a reduced ability to chemotact toward cyclic AMP in the presence of exogenous
PDE. The results also suggest that the cyclic AMP receptor is not a glycoprotein
although it has to be borne in mind that certain glycoproteins still retain biological activity in their non-glycosylated states (Speake et al. 1981; Onishi et al.
1979). The reversibility of the inhibition of development by TM may be useful
in studies of the reaggregation of cells rendered non-cohesive by the TM inhibition of expression of cohesion-mediating molecules both in Dictyostelium
and in other cell types.
We would like to thank the SERC forfinancialsupport in the form of a project grant and
a studentship to C.J.M. We also thank Mrs Jane Drew for her excellent technical assistance.
REFERENCES
ALCANTARA, F. & MONK, M. (1974). Signal propogation during aggregation in the slime mould
Dictyostelium discoideum. J. gen. Microbiol. 85, 321-334.
BEUG, H., KATZ, F. E. & GERISCH, G. (1973). Dynamics of antigenic membrane sites relating
to cell aggregation in Dictyostelium discoideum. J. Cell Biol. 56, 647-658.
BONNER, J. T. (1944). A descriptive study of the development of the slime mould Dictyostelium discoideum. Amer. J. Bot. 31, 175-182.
BONNER, J. T. (1949). The demonstration of acrasin in the later stages of the development of
the slime mould Dictyostelium discoideum. J. exp. Zool. 110, 259-271.
BONNER, J. T. (1963). Epigenetic development in the cellular slime molds. Symp. Soc. exp.
Biol. 17, 341-358.
BONNER, J. T. (1971). Aggregation and differentiation in the cellular slime molds. Ann. Rev.
Microbiol. 25, 75-92.
DARMON, M. & KLEIN, C. (1978). Effects of amino acids and glucose on adenylate cyclase and
cell differentiation of Dictyostelium discoideum. Devi. Biol. 63, 377-389.
DARMON, M., BARRA, J. & BRACHET, P. (1978). The role of phosphodiesterase in aggregation
of Dictyostelium discoideum. J. Cell Sci. 31, 233-243.
Tunicamycin and the development of Dictyostelium
247
E. & GERISCH, G. (1977). Implications of developmentally regulated concanavalin A
binding proteins of Dictyostelium in cell adhesion and cyclic AMP regulation. Cell Differentiation. 6, 339-346.
FRANKE, J. & KESSIN, R. H. (1981). The cyclic nucleotide phosphodiesterase inhibitory
protein of Dictyostelium discoideum. Purification and characterisation./. biol. Chem. 256,
7628-7637.
GERISCH, G. (1968). Cell aggregation and differentiation in Dictyostelium. Curr. Topics devl.
Biol. 3, 159-197.
GERISCH, G. (1979). Control circuits in cell aggregation and differentiation of Dictyostelium
discoideum. In Mechanisms of Cell Change (eds J. D. Ebert & T. S. Okada) pp. 225-239.
New York: John Wiley & Sons.
GERISCH, G., MALCHOW, D., RIEDEL, V., MULLER, E. & EVERY, M. (1972). Cyclic AMP-phosphodiesterase and its inhibitor in slime mould development. Nature New Biol. 235,90-92.
GINGLE, A. R. (1977). cAMP enhanced aggregation of cells from early chick embryos. Devi
Biol. 58, 394-401.
GOIDL, E. A., CHASSY, B. M., LOVE, L. L. & KRICHEVSKY, M. (1972). Inhibition of aggregation and differentiation of Dictyostelium discoideum by antibodies against cyclic AMP
phosphodiesterase. Proc. natnAcad. ScL, U.S.A. 69,1128-1130.
GREEN, A. A. & NEWELL, P. C. (1975). Evidence for the existence of two types of cAMP
binding sites in aggregating cells of Dictyostelium discoideum. Cell 6, 129-136.
GUILLERMET, C. & MANDARON, P. (1980). In vitro imaginal disc development and moulting
hormone. /. Embryol. exp. Morph. 57, 107-118.
HENDERSON, E. J. (1975). The cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum: Binding characteristics of aggregation-competent cells and variation of
binding levels during the life cycle. /. biol. Chem. 250, 4730-4736.
KESSIN, R. H., ORLOW, S. J., SHAPIRO, R. I. & FRANKE, J. (1979). Binding of inhibitor alters
kinetic and physical properties of extracellular cyclic AMP phosphodiesterase from
Dictyostelium discoideum. Proc. natnAcad. ScL, U.S.A. 76, 5450-5454.
KLEIN, C. & DARMON, M. (1977). Effects of cAMP pulses on adenylate cyclase and the
phosphodiesterase inhibitor of D. discoideum. Nature 268, 16-11.
KONIJN, T. M. (1972). Cyclic AMP as first messenger. In Advances in Cyclic Nucleotide
Research Vol. I, pp. 17-31. New York: Raven Press.
EITTLE,
KONIJN, T. M., VAN DE MEENE, J. G. C , CHANG, Y. Y., BARKLEY, D. S. & BONNER, J. T.
(1969). Identification of adenosine-3', 5'-monophosphate as the bacterial attractant for
myxamoebae of Dictyostelium discoideum. J. Bad. 99, 510-512.
LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227, 680-685.
LAM, T-Y. & Sui, C-H. (1982). Inhibition of cell differentiation and cell cohesion by
tunicamycin in Dictyostelium discoideum. Devi Biol. 92, 398-407.
LENNARZ, W. J. ed. (1980). The Biochemistry of Glycoproteins and Proteoglycans, New York:
Plenum Press.
LOOMIS, W. F. (1975). "Dictyostelium discoideum: A Developmental System." New York:
Academic Press.
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. K. & RANDALL, R. J. (1951). Protein measurement with Folin phenol reagent. /. biol. Chem. 193, 265-275.
MALCHOW, D. & GERISCH, G. (1974). Short-term binding and hydrolysis of cyclic 3', 5'
adenosine monophosphate by aggregating Dictyostelium cells. Proc. natn Acad. Sci.,
U.S.A. 71, 2423-2427.
MALCHOW, D., NAGELLE, B., SCHWARZ, H. & GERISCH, G. (1972). Membrane-bound cyclic
AMP phosphodiesterase in chemotactically responding cells of Dictyostelium discoideum.
Eur. J. Biochem. 28, 136-142.
MARIN, F. T., BOYETTE-BOULAY, M. & ROTHMAN, F. G. (1980). Regulation of development
in Dictyostelium discoideum: III. Carbohydrate-specific intracellular interactions in early
development. Devi Biol. 80, 301-312.
MULLER, K. & GERISCH, G. (1978). A specific glycoprotein as the target site of adhesion
blocking Fab in aggregating Dictyostelium cells. Nature 274, 445-449.
248
C. J. MCDONALD AND J. SAMPSON
K. D. & HOMEWOOD, C. A. (1974). Introduction to Liquid Scintillation Counting,
London: Butterworths.
NEWELL, P. C. (1978). Cellular communication during aggregation of Dictyostelium, the
Second Fleming Lecture. /. gen. Microbiol. 104, 1-13.
OAKLEY, B. R., KIRSCH, D. R. & MORRIS, N. R. (1980). A simplified ultrasensitive silver stain
for detecting proteins in polyacrylamide gels. Anal. Biochem. 105, 361-363.
ONISHI, H. R., TKACZ, J. S. & LAMPEN, J. O. (1979). Glycoprotein nature of yeast alkaline
phosphatase. Formation of active enzyme in the presence of tunicamycin. /. biol. Chem.
254, 11943-11952.
ORLOW, S. J., SHAPIRO, R. I., FRANKE, J. & KESSIN, R. H. (1981). The extracellular cyclic
AMP phosphodiesterase of Dictyostelium discoideum: purification and characterisation. J.
biol. Chem. 256, 7620-7627.
PANNBACKER, R. G. & BRAVARD, L. J. (1972). Phosphodiesterase in Dictyostelium discoideum
and the chemotactic response to cyclic adenosine monophosphate. Science 175,1014-1015.
RIEDEL, V., GERISCH, G., MULLER, E. & BEUG, H. (1973). Defective cyclic adenosine-3',5'phosphate phosphodiesterase regulation in morphogenetic mutants of Dictyostelium
discoideum. J. molec. Biol. 74, 573-585.
ROBERTSON, A. D. J. & GRUTSCH, J. F. (1981). Aggregation in Dictyostelium discoideum. Cell
14, 603-611.
ROSEN, S. D., KAFKA, J. A., SIMPSON, D. L. & BARONDES, S. H. (1973). Developmentallyregulated carbohydrate-binding proteins in Dictyostelium discoideum. Proc. natn Acad.
Sci, U.S.A. 70, 2554-2557.
SAMPSON, J., TOWN, C. D. & GROSS, J. (1978). Cyclic AMP and the control of aggregative
gene expression in Dictyostelium discoideum. Devi Biol. 67, 54-64.
SPEAKE, B. K., MALLEY, D. J. & HEMMING, F. W. (1981). The effect of tunicamycin on
secreted glycosides of Aspergillus niger. Arch. Biochem. Biophys. 210, 110-117.
TAKATSUKI, A., ARIMA, K. & TAMURA, G. (1971). Tunicamycin, a new antibiotic. I. Isolation
and characterisation of tunicamycin. /. Antibiot. 1A, 215-233.
TKACZ, J. S. & LAMPEN, J. O. (1975). Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf liver microsomes. Biochem. Biophys. Res. Commun. 65, 248-257.
TODA, K., ONO, K-I. & OCHIAI, H. (1980). Surface labelling of membrane glycoproteins and
their drastic changes during development of Dictyostelium discoideum. Eur. J. Biochem.
I l l , 377-388.
WATTS, D. J. & ASHWORTH, J. M. (1970). Growth of myxamoebae of the cellular slime mould
Dictyostelium discoideum in axenic culture. Biochem. J. 119, 171-174.
YAMADA, H., HIRANO, T., MIYAZAKI, T., TAKATUKI, A. & TAMURA, G. (1982). Effects of
tunicamycin on cell adhesion and biosynthesis of glycoproteins in aggregation competent
cells of Dictyostelium discoideum. J. Biochem. (Tokyo) 92, 399-407.
NEAME,
{Accepted 18 August 1983)