/. Embryo!, exp. Morph. Vol. 48, pp. 153-160, 1978
Printed in Great Britain © Company of Biologists Limited 1978
153
Participation of the cell surfaces
in determining the developmental courses in the
cellular slime mould Dictyostelium purpureum
By M. SAITO 1 AND K. YANAGISAWA 2
From the Department of Biological Sciences, University of Tsukuba
SUMMARY
Dictyostelium purpureum S5 and S6, mating type strains, form fruiting-bodies in a monoclonal culture, but produce macrocysts in a mix culture. The effects of Concanavalin A
(Con A) on both fruiting-body formation and macrocyst formation, and changes of Con Amediated cell agglutinability during development were studied. It was found that Con A
inhibits macrocyst formation but not fruiting-body formation, and that macrocyst-forming
cells are much more susceptible to Con A agglutination than are fruiting-body-forming cells
during the aggregation stages. When fruiting-body-forming cells are treated with either
trypsin or a-chymotrypsin, their Con A agglutinability is enhanced to the same extent as
that of macrocyst-forming cells. It was also found that when S6 cells are treated with proteases they sometimes produce normal macrocysts even in a monoclonal culture.
The results obtained in these experiments showed that the surface properties of fruitingbody-forming cells and macrocyst-forming cells are different, and that the cell surface might
play an important role in determining the two developmental courses.
INTRODUCTION
Cellular slime moulds have two modes of development. One is fruiting-body
formation and the other is macrocyst formation. When foods are exhausted,
myxamoebae aggregate and form a pseudoplasmodium which is subsequently
transformed into a fruiting-body composed of spore and stalk cells (Bonner,
1959). Under certain conditions, however, cells form a macrocyst; a multicellular mass encased in a loose fibrillar sheath (Blaskovics & Raper, 1957;
Filosa & Dengler, 1972; Erdos, Nickerson & Raper, 1972; Nickerson & Raper,
1973). Macrocyst formation also begins with aggregation of myxamoebae in the
same way as that of fruiting-body formation.
There is good cytological and genetical evidence to indicate that the macrocyst
represents the true sexual phase in cellular slime moulds (Macinnes & Francis,
1
Author's address: Department of Biology, Teikyo University, Kanagawa 199-01, Japan.
Author's address (for reprints): Department of Biological Sciences, University of Tsukuba,
Ibaraki 300-31, Japan.
2
154
M. SAITO AND K. YANAGISAWA
1974; Francis, 1975), while the fruiting-body represents the non-sexual phase.
In some species, such as Dictyostelium mucoroides, the macrocyst can be formed
in a monoclonal culture (Filosa, 1962). In most species, however, macrocysts
are only produced when cells of two strains of the mating type are mixed
(Erdos, Raper & Vogen, 1973; Clark, Francis & Eisenberg, 1973; Francis,
1973). Dictyostelium purpureum requires also mix of cells of two strains of the
mating type to produce the macrocysts.
We studied effects of Con A and proteases on development and differentiation,
and changes of the cell surface properties during the aggregation stages of
development in D. purpureum by using Con A. The results showed that the cell
surfaces play an important role in determining the two developmental courses
of cellular slime mould.
MATERIALS AND METHODS
D. purpureum, strains S5 and S6 were used in this experiment. These were
kindly supplied by Dr T. Yamada, Tokyo Metropolitan Isotope Centre, who
had isolated them from the soil of Suwa, Japan. The cells of these strains form
fruiting-bodies when they are cultured separately, but produce macrocysts when
they are mixed and incubated in the dark. The myxamoebae were grown in
association with Aerobacter aerogenes on nutrient agar plates (Sussman, 1966).
To form fruiting-bodies, vegetative amoebae of S5 and S6 were harvested,
washed three times by centrifugation at 1500 rev./min for 6 min, and suspended
in a buffered salt solution (Newell, Telser & Sussman 1966) at a density of
2 x 107 amoebae/ml. Small amounts (0-5-0-8 ml) of the suspensions were placed
on a lactose peptone (LP) agar plate (9 cm in diameter) separately, and
incubated at 22 °C for 3 days in the dark or in light.
To form macrocysts, the suspended amoebae of S5 and of S6 were mixed.
Five millilitres of the mixture were placed on the LP agar and incubated at
25 °C for 3 days in the dark (Nickerson & Raper, 1973).
In order to examine the effects of Con A on the formation of fruiting-bodies
and of macrocysts, the suspended amoebae were placed on LP agar plates
containing 110/miole/ml NaCl and different concentrations of Con A (10300 /tg/ml) dissolved in the agar.
Con A-mediated cell agglutinability during development was assayed according to the method described by G. Weeks (1973). The cells of the different stages
of development were taken from fruiting-body-forming plates and macrocystforming plates. After dissociation of the cells by pipetting, they were suspended
in Bonner's salt solution (Bonner, 1947) at a density of 2x 106 cells/ml. Then
Con A was added to the cell suspension at final concentrations of 3, 40 and
100 /tg/ml. The suspensions were shaken on a reciprocal shaker at 120 cycles
per min at 22 °C. After 10 min of shaking, aliquots of the suspension were
withdrawn and the number of unagglutinated cells was counted under a micro-
Cell surface in the development of Dictyostelium
155
Table 1. Effects of Con A on the formation of macrocysts
,
0
Concentrations of Con A (/tg/ml)
*
10
50
70
100
150
200
,
300
Addition of 005 M of
+
+
+
+
+
+
+
a-2-methyl-mannoside
+ and ± , Formation of macrocysts and of pseudomacrocysts, respectively.
—, No formation of either macrocysts or pseudomacrocysts.
scope. The degree of Con A-mediated cell agglutinability was calculated from
the following formula,
m
(no. of cells as singles + no. of cells as doubles) Con A
(no. of cells as singles + no. of cells as doubles) control
m
Since even in the absence of Con A, a significant number of cells are always
observed as doublets, cells viewed as doublets were counted as unagglutinated.
The effects of trypsin and a-chymotrypsin on Con A-mediated cell agglutinability during development were tested by the following methods. After
different periods of incubation, the cells of S5 and S6 were taken, dissociated
and suspended in the buffered salt solution, then treated with either 100/tg/ml
of trypsin or a-chymotrypsin for 20 min at 22 °C. A Con A-mediated agglutination assay was conducted after the cells were washed and suspended in
Bonner's salt solution.
In order to examine the effects of the proteases on differentiation, theproteasetreated cells of S5 and S6 were placed on LP agar plates separately, and incubated
for 4 days at 25 °C in the dark, then the presence or absence of macrocysts was
examined.
RESULTS
Effects of Con A on the formation of fruiting-bodies
After 3 days of incubation either in the dark or in light, the cells of S5 and S6
aggregated and formed normal fruiting-bodies on the plates containing Con A.
Development was not ceased by any of concentrations of Con A that were
tested, although a delay in aggregation was observed. The delay is dependent
of the concentrations of Con A. It was about 6 h at the concentration of
30 /tg/ml Con A and 8 h at 70
Effects of Con A on the formation of macrocysts
The result was markedly different from that of the formation of fruitingbodies. After 3 days of incubation in the dark, normal macrocysts were produced on the agar plates containing Con A at less than 50 /tg/ml. The process
156
M. SAITO AND K. YANAGISAWA
10
Fig. 1. Changes of Con A-mediated cell agglutinability during development.
Abscissa: time in development (h); Ordinate: degree of Con A-mediated agglutinability. # , S5 and S6 cells incubated in the dark which are forming macrocysts;
x, +, S5, S6 cells which are forming fruiting-bodies, respectively; O, S5 and S6
cells incubated in light which are forming fruiting-bodies; A, a-2-methyl mannoside
was added to a S5 and S6 cell culture incubated in the dark.
of the formation of macrocysts was also normal, although a delay in aggregation
was seen. On the plates containing Con A at 70 /*g/ml, however, normal macrocysts were not formed. Pseudomacrocysts: the cell clusters bounded with
a single thin membrane, and cell clumps were formed. At concentrations higher
than 100/tg/ml, only cell clumps were formed, even after 4 days of incubation.
These results are shown in Table 1.
The inhibitory effect of Con A on macrocyst formation is reversible. When the
cell clumps which could not produce macrocysts were harvested, washed,
dissociated and placed on LP agar plates containing no Con A. Normal macrocysts were formed after continuous incubation of 3 days in the dark. The
inhibitory effect of Con A was completely abolished when a-2-methyl mannoside,
a competitive inhibitor of Con A binding, was added to the plates at a final
concentration of 5 x 10~2 M.
Changes of Con A-mediated cell agglutinability during development
Con A-mediated cell agglutinability changes during development. It increased
for the first 5 h during development in both fruiting-body-forming cells and
macrocyst-forming cells. After 5 h, it decreased in fruiting-body-forming cells,
but remained constant in macrocyst-forming cells. Macrocyst-forming cells
were more readily agglutinated by Con A than were fruiting-body-forming cells
during all stages of development which were tested. Figure 1 indicates the
changes of Con A-mediated cell agglutinability (3 /*g/ml of Con A) during
development.
Cell surface in the development of Dictyostelium
157
100
10
Fig. 2. Increase of Con A-mediated cell agglutinability after proteolytic enzymetreatment. Abscissa: time in development (h); Ordinate: degree of Con A-mediated
agglutinability. # , S5 and S6 cells which are forming macrocysts; + , S6 cells
which are forming fruiting-bodies; • , A, S6 cells which are treated with a-chymotrypsin and trypsin, respectively.
Similar results were also obtained when high concentrations of Con A
(40 & 100/fcg/ml) were used.
When 5 x 10~2 M of a-2-methyl mannoside was added at a final concentration
to the cell suspensions containing 100 fig/ml of Con A, the cells showed same
agglutinability with Con A that the control cells did (Fig. 1).
Effects of proteases on Con A-mediated cell agglutination
It is known that proteases change the nature of cell surfaces (Nicolson, 1972;
Hynes, 1974; Hoffman & McMahon, 1977; Jermyn, Kilpatrick, Schmidt &
Stirling, 1977). In mammalian cells, for example, protease-treated cells are more
readily agglutinated by Con A than are untreated cells. We, therefore, suspected
that if fruiting-body-forming cells were treated with proteases, they might
agglutinate to the same extent as macrocyst-forming cells do.
In order to test this possibility, singly cultured S6 cells from different stages
of development were dissociated and treated with either trypsin or a-chymotrypsin, after which a Con A-mediated agglutination assay (3 /*g/ml of Con A)
was performed. The enzyme-treated cells showed a much higher agglutinability
with Con A than the control cells did. The degree of Con A-mediated
agglutination in the pro tease-treated cells was about the same as that of the
macrocyst-forming cells (Fig. 2).
EMB 48
158
M. SAITO AND K. YANAGISAWA
Effects of the proteases on cell differentiation
The experiments described above demonstrated that macrocyst-forming cells
are more readily agglutinated by Con A than are fruiting-body-forming cells,
and that fruiting-body-forming cells treated with the proteases are agglutinated
by Con A to the same extent as macrocyst-forming cells. This evidence
suggests that S5 and S6 cells possibly can produce macrocysts after proteolysis
even in a monoclonal culture. The cells of S5 and S6 of different stages (0-10 h
of development) were collected, dissociated and placed on LP agar plates
separately after proteolysis. After 4 days of incubation in the dark, the presence
of macrocysts was examined. S5 cells in a single culture produced no macrocysts,
while S6 cells taken from 6-8 h stages of development formed sometimes a few
normal macrocysts even in a single culture. In nine plates out of 80 which were
examined, two to ten normal macrocysts were found.
DISCUSSION
The present studies showed that macrocyst formation in D. purpureum is
totally inhibited by the concentrations higher than 100/^g/ml of Con A.
However, fruiting-body formation is not ceased even by 300 /tg/ml of Con A.
This evidence shows that the surface properties of macrocyst-forming cells may
be different from those of fruiting-body forming cells. It suggests also the
possibility that there are some Con A binding sites in the cell surfaces which are
specifically involved in the process of macrocyst formation. According to West
& McMahon (1977) there are more than 35 different kinds of Con A receptors
in the plasma membrane of D. discoideum. Probably D. purpureum has a large
number of con A binding sites in the plasma membrane. At present however,
we don't know which sites may be actually involved in the process of the
formation of macrocysts. These should wait for further studies in future. Assays
of Con A-mediated cell agglutination showed clearly that cell surface properties
of macrocyst-forming cells are different from those of fruiting-body-forming
cells. It also revealed that Con A-mediated cell agglutinability changes during
development, and the changing pattern of macrocyst-forming cells is dissimilar
to that of fruiting-body-forming cells. The mechanism of changes in Con Amediated cell agglutinability during development is unclear at present, although
there are several explanations. It cannot be explained, however, by alteration in
the number of receptors, because the number of receptors per cell shows little
changes during development in cellular slime moulds (Weeks, 1975; Kawai &
Takeuchi, 1976). A more plausible explanation may be that some kinds of
modification or replacement of the original Con A binding sites take place
during development, thereby producing changes in the agglutinability (Weeks,
1975). It is a fact that in D. discoideum many Con A receptors on the cell surface
of the vegetative stage disappear and new sites appear during the course of
Cell surface in the development of Dictyostelium
159
development. In addition, many of the remaining receptors alter their affinity
to Con A with time (West & McMahon, 1977). There is another possible
explanation, however, to account for the changes of Con A-mediated cell
agglutinability. Gillette, Dengler & Fillosa (1974) found occurrence of redistribution of Con A binding sites on the cells of D. discoideum. Changes in
membrane fluidity permitting redistribution of the surface receptors may
produce changes in Con A-mediated cell agglutinability.
It is well known that in mammals the cell agglutinability can be increased by
proteolyses (Nicolson, 1972; De Petris, Raff& Mallucci, 1973). When fruitingbody-forming cells were treated with the proteases, the agglutinability was
enhanced to the same extent as that of macrocyst-forming cells. The increase of
Con A-mediated cell agglutinability by the treatement of the proteases may be
explained by changes either in membrane components or in surface mobility,
although the real nature of the enhancement of the agglutinability is also obscure
at present.
It is most interesting that S6 cells can produce a macrocyst without S5 cells
after the proteolytic enzyme-treatment. This fact suggests that the cell surface
may be involved in determining the two developmental courses in the cellular
slime mould. It is, however, that after proteolysis only S6 cells and not S5 cells
make macrocysts in a monoclonal culture. This could be due to some differences
in the cell surface properties between these two mating type cells.
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{Received 17 April 1978, revised 10 August 1978)