Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
163
CHEMOTAXIS IN DICTYOSTELIUM DISCOIDEUM: EFFECT OF CONCANAVALIN A ON CHEMOATTRACTANTMEDIATED CYCLIC GMP ACCUMULATION AND LIGHT SCATTERING DECREASE Josh M. Mato,
Peter J.M. van Haastert, and Theo M. Konijn
Cell Biology and Morphogenesis University of Leiden, Leiden,
Frans
A. Krens
Unit, Kaiserstraat The Netherlands
63,
ABSTRACT In cells of the cellular slime mold Dictyostelium discoideum concanavalin A (Con A), at a concentration of 100 pg per ml, inhibits folic acid and cyclic AMP induced decrease in light scattering. Con A has no effect on folic acid mediated cyclic GMP accumulation and increases cyclic AMP mediated cyclic GMP accumulation two-fold. At a lower Con A concentration, 10 ug per ml, changes in light scattering induced by folic acid are normal and cyclic AMP induces a monophasic instead of a biphasic response. The stimulatory effect of Con A on cyclic AMP mediated cyclic GMP accumulation is still observable at 10 ug Con A per ml. When cells are repeatedly stimulated with cyclic AMP, a decrease in light scattering without being accompanied by changes in cyclic GMP concentration is observed. Based on these results a model for chemotaxis is proposed. INTRODUCTION Vegetative cells of Dictyostelium discoideum are attracted by folic acid (Pan et al., 1972) and aggregative cells by cyclic AMP (Konijn et al., 1968). Both chemoattractants induce in cell suspensions of sensitive cells a fast increase of cyclic GMT?(Mato et al., 1977a and b; Wurster et al., 1977a) and a decrease of light scattering (Gerisch and Hess, 1974; Wurster and Schubiger, 1977). In D.lacteum and Polysphondilium violaceum cyclic GMP levels also increase in response to their specific attractants (Mato and Konijn, 1977; and Wurster et al., 1977b). In the present study we have investigated the role of concanavalin A (Con A) on attractant mediated cyclic GMP accumulation and light scattering decrease in cell suspensions of D.discoideum. Con A is known to increase the binding of cyclic AMP to the cellsurface of D.discoideum (Juliani and Klein, 1977) and to inhibit the chemotactic response to this attractant (Gillette and Filosa, 1973). Con A receptors have been visualized on the cell-surface of cells of D.discoideum (Molday et al., 1976) and a developmentally regulatedcarbohydrate binding protein, discoidin, has been purified from this species (Rosen et al., 1973).
164
Cell Biology
International
vegetative
aggregat
stage
stage
Reports,
Vol. 2, No. 2, 1978
ive
Control
Con A
++,,,-
100t.dml
Con
A
10 w/ml
7CAMP
f FA
/’
t FA
L *
TcAMP
Fig.1. Changes in light scattering in cell suspensions of D.discoideum in response to folic acid and cyclic AMP in the presence and absence of Con A. Cells were starved for 1 hour (vegetative stage) or for 5 hours (aggregative stage). When indicated by the arrow cells were stimulated with 3x10m6M folic acid (FA) or 3x10-k cyclic AMP.
Cell Biology International
Reports, Vol. 2, No. 2, 1978
165
RESULTS Effect of Con A on attractant induced decrease of light scattering. Incubation of cells of D.discoideum for 10 minutes with IO0 pg Con A per ml inhibits folic acid and cyclic AMP induced changes in light scattering completely (fig. 1). After 20 minutes acid inducincubation in the presence of 10 pg Con A per ml folic ed a normal decrease of light scattering (fig. 1). In contrast to the control, cells treated with 10 ug Con A per ml respond to cyclic AMP with a monophasic decrease in light scattering (fig. 1). After 10 minutes in the presence of 100 pg Con A per ml cells form fewer and smaller clumps than in the control, while in the presence of 10 ug Con A per ml the size and number of clumps is similar to that observed in the controls. In the presence of 1 mM EGTA cells also form very few and small clumps yet, they show a normal light scattering decrease in response to folic acid and cyclic AMP (fig. 2). This indicates that the inhibitory effect of Con A on light scattering is not due to differences in clump formation. The Con A ligand, a-D-glycopyranoside, counteracts the effect of Con A on light scattering (data not shown). Effect of Con A on attractant induced cyclic GMF' accumulation. In the presence of 100 ug Con A per ml vegetative cells of D.discoideum respond to folic acid with normal changes in the cellular content of cyclic GMP (fig. 3a). Contrary to what happens during the vegetative stage, aggregative cells treated with 100 ug Con A per ml respond to cyclic AMP with an increase in cyclic GMP double than that observed in the control (fig. 3b). The effect of Con A on cyclic AMP induced cyclic GMP accumulation is still observable at 10 pg Con A per ml (fig. 3b). Effect of cyclic AMP on light scattering and cyclic GMP when cells are triggered repeatedly. Fig. 4 shows the changes in light scattering and cyclic GNP when aggregative cells of D.discoideum are triggered repeatedly with cyclic AMP. A normal decrease in light scattering is induced by a second cyclic AMP pulse when given 30 seconds after a cyclic AMP pulse of the same magnitude. Contrary to what happens with light scattering, a second cyclic AMP pulse, only induces a very small, if any, change in the cyclic GMP content. DISCUSSION In D.discoideum incubation of cells for 5 minutes with 100 ug Con A per ml increases about twice the binding of cyclic AMF' to cell-surface receptors (Juliani and Klein, 1977). The present work indicates that the increased cyclic AMP binding in the presence of Con A is translated into an enhanced cyclic GMP accumulation in response to cyclic AMF'. Apparently, Con A unmasks functional receptors and did not induce unspecific binding. The Con A effect on atis developmentally regtractant mediated cyclic GMP accumulation ulated. This is shown by the lack of effect of Con A on folic acid
166
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
mediated cyclic GMP accumulation. A carbohydrate-binding protein has been purified from aggregative cells of D.discoideum (Rosen et al., 1973). The possibility that this protein activates attractant mediated cyclic GMP accumulation is under investigation. If so, this protein may not only be involved in cell to cell adhesion but also in the detection of a chemotactic signal. The inhibition by Con A of attractant induced changes in light scattering may also explain the decreased chemotaxis observed in D.discoideum in the presence of this lectin (Gillette and Filosa, 1973).
d
t FA
%4J t CAMP
Fig. 2. Changes in light scattering in cell suspensions of D.discoideum in the presence of 1 mM EGTA in response to folic acid and cyclic AMP. After starvation cells were washed twice in cold 10 mM phosphate buffer containing 1 mM EGTA, pH adjusted to 6.0, and resuspended in this same buffer at a cell density of 1x10* cells per ml. When indicated by the arrow vegetative cells (1 hour starvation) were triggered with 3x10e6M folic acid (FA) and aggregative cells (5 hours starvation) with 3x10-*M cyclic AMP.
Cell Biology
International
Reports,
167
Vol. 2, No. 2, 1978
We have previously shown (Mato et al., 1977a) that after triggering cells with 5x10m8M cyclic AMP a refractory period of 1 minute is induced. In contrast to these results, normal changes in light scattering are induced by a second cyclic AMP pulse when These results argue against a posgiven 30 set after the first. sible role of cyclic GMP in the generation of changes in light scattering. The results with Con A show that it is also possible to get changes in cyclic GMP without modifying the light scattering. Therefore, it is concluded that the interaction cyclic AMPa decrease in light scattering receptor(s) generates two signals: scattering and an increase in cyclic GMP. The decrease in light may be due to cell contraction (Wurster et al., 3977b). As shown is independent of by the experiments with EGTA, this contraction the presence of extracellular calcium (see also Gerisch et al., 1975).
7, a
14-
5,
10,
3-
6,
l-
21
0
L
20
I
I
40
60
1
Sec.
b
1
1
0
20
I
40
60
sec.
Fig. 3. Changes in cyclic GM? content in cell suspensions of D.discoideum in response to folic acid and cyclic AMP in the presence and absence of Con A.(a) Cells starven for I hour were stimulated at time zero with 3x10m6M folic acid: (0) control; and (0) preincubated for 10 minutes with 100 ug Con A per ml (b) Cells starven for 5 hours were stimulated at time zero with 3x10-* M cyclic AMP: (0) control; (4) preincubated for 20 minutes with 10 ng Con A per ml; and (0) preincubated for 10 minutes with 100 pg Con A per ml.
168
Cell Biology
International
Reports,
Vol. 2, No. 2, 7978
Cyclic GMPaccumulation in D.discoideum is also independent of extracellular calcium (Mato et al., 1977a) and in smooth muscle cyclic GMP accumulation has been correlated with cellular relaxation (Schultz et al., 1977). Based on these results we propose that chemotaxis in D.discoideum is the result of the following two processes: i) contraction over the whole cell, e.g. triggered by the release of calcium from intracellular deposits; and ii) formation of an intracellular gradient of cyclic GMPwhich causes the extension of a pseudopod by means of relaxation of the anterior part of the cell.
4
.
2 3 .I
0
-/
t
1
0
0.5
1
1.5
2
mi n.
T T Fig. 4. Effect of cyclic AMP on light scattering and cyclic GMP content when cells of D.discoideum are stimulated repeatedly. Cells starven for 5 hours were stimulated at time zero with 3x10-sM cyclic AMP and for a second time, with a pulse of the same magnitude, 30 set later.
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
169
MATERIALS AND METHODS D.discoideum, NC-4(H), was used for all experiments. Cells were grown and harvested as described by Konijn and Raper (1961). After centrifugation cells were resuspended in 10 mM phosphate buffer of lo7 cells per ml. Starvation was induced pH 6.0, at a density by shaking (Gerisch, 1962). After shaking, cells were centrifuged, washed twice in cold phosphate buffer, and adjusted to 1~10~ cells per ml. One portion of the suspension was then used for light scattering measurements and another portion for cyclic GMP determinations. Light scattering was recorded as described by Gerisch and Hess (1974) except that cells were aerated instead of oxygenated. For measuring the optical density at 405 nm a Beckman Acta III spectrophotometer was used. Cyclic GMP measurements were carried out as previously described (Mato et al., 1977 a and b). Material for the radioimmunoassay of cyclic GMP was provided by Amersham. Results are given as pmoles cyclic GMP per IO7 cells. Cyclic GMP and cyclic AMP were purchased from Sigma. Concanavalin A, was from Calbiochem.
from Boehringer. A, lyophilized
Folic acid was in NaCl, grade
ACKNOWLEDGMENTS The technical acknowledged.
assistance
of Miss
A. van Oosten
is gratefully
REFERENCES Gerisch, G. (1962) Zellfunktionen und Zellfunktionswechsel in der Entwicklung von Dictyostelium discoideum. IV. Der Zeitplan der Entwicklung. Wilhelm Roux Archiv fur Entwicklungsmechanik, -f153 603-520. oscillations Gerisch, G. and Hess, B. (1974) Cyc 1ic AMP controlled in suspended Dictyostelium cells: Their relation to morphogenetic cell interactions. Proceedings of the National Academy of Sciences USA, 71, 2118-2122. Gerisch, G., Malchow, D., Huesgen, A., Nanjundiah, V., ROOS, W. and Wick, U. (1975) Cyclic AMP reception and cell recognition in Dictyostelium discoideum. In: D.McMahon and C.F. Fox (eds.). Developmental Biology, ICN-UCLA symposia on Molecular and Cellular Biology. Vo1.2, pp.76-88. W.A. Benjamin, Inc. Gillette, M.U. and Filosa, M.F. (1973) Effect of Concanavalin A on cellular slime mold development: premature appearance of membrane-bound cyclic AMP phosphodiesterase. Biochemical and Bipphysical Research Communications, 53, 1159-1166. Juliani, M.H. and Klein, C. (1977) Calcium ion effects on cyclic adenosine 3':5' monophosphate bindings to the plasma membrane of Dictyostelium discoideum. Biochimica et Biophysics Acta, 497, 369-376.
170
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
in DictyosteKonijn, T.M. and Raper, K.B. (1961) Cell aggregation lium discoideum. Develop. Biol.,?, 725-756. Konijn, T.M., Barkley, D.S., Chang, Y.Y. and Bonner, J.T. (1968) occurring acrasin in the cellular slime Cyclic AMP: a naturally molds. American Naturalist, 102, 22.5-234. Mato, J.M., Krens, F.A., van Haastert, P.J.M. and Konijn, T.M. (1977a) Cyclic AMP dependent cyclic GMP accumulation in Dictyostelium discoideum. Proceedings of the National Academy of Sciences USA, 74, 2348-2351. P.J.M., Krens, F.A., Rhijnsburger, E.H., Mato, J.M., van Haastert, T.M. (1977b) Cyclic AMP and folic Dobbe, F.C.P.M. and Konijn, acid mediated cyclic GMP accumulation in Dictyostelium discoideum. FEBS Letters, 2, 331-336. Mato, J.M. and Konijn, T.M. (1977) The chemotactic signals and cyclic GMP accumulation in Dictyostelium. In: P. Cappuccinelli and J.M. Ashworth (eds.). Proceedings of the EMBO-workshop on Development and Differentiation in Cellular Slime Moulds. Vol.], pp.93-103. Elsevier, North-Holland. R. and McMahon, D. (1976) Concanavalin A and Molday, R., Jaffe, wheat germ agglutinin receptors on Dictyostelium discoideum. The Journal of Cell Biology, 71, 314-322. E.M. and Bonner, J.T. (1972) Folic acid as second Pan, P., Hall, chemotactic substance in the cellular slime moulds. Nature New Biology, 237, 181-182. Rosen, S.D., Kafka, J.A., Simpson, D.L. and Barondes, S.H. (1973) Developmentally regulated carbohydrate binding protein in Dictyostelium discoideum. Proceedings of the National Academy of Sciences USA, 70, 2554-2557. Schultz, K.D., Schultz, K. and Schultz, G. (1977) Sodium nitroprusside and other smooth muscle-relaxants increase cyclic GMP levels in rat ductus deferens. Nature, 265, 750-751. Wurster, B., Schubiger, K., Wick, U. and Gzsch, G. (1977a) Cyclic GMP in Dictyostelium discoideum: oscillations and pulses in response to folic acid and cyclic AMP signals. FEBS Letters, 76, 141-144. Wurster, B., Bozarro, S. and Gerisch, G. (1977b) Cyclic GMP regulation and responses of Polysphondylium violaceum to chemoattractants. Cell Biology, in press. Wurster, B. and Schubiger, K. (1977) Oscillations and cell development in Dictyostelium discoideum stimulated by folic acid pulses. Journal of Cell Science, in press.
International
Reports,
Vol. 2, No. 2, 1978
163
CHEMOTAXIS IN DICTYOSTELIUM DISCOIDEUM: EFFECT OF CONCANAVALIN A ON CHEMOATTRACTANTMEDIATED CYCLIC GMP ACCUMULATION AND LIGHT SCATTERING DECREASE Josh M. Mato,
Peter J.M. van Haastert, and Theo M. Konijn
Cell Biology and Morphogenesis University of Leiden, Leiden,
Frans
A. Krens
Unit, Kaiserstraat The Netherlands
63,
ABSTRACT In cells of the cellular slime mold Dictyostelium discoideum concanavalin A (Con A), at a concentration of 100 pg per ml, inhibits folic acid and cyclic AMP induced decrease in light scattering. Con A has no effect on folic acid mediated cyclic GMP accumulation and increases cyclic AMP mediated cyclic GMP accumulation two-fold. At a lower Con A concentration, 10 ug per ml, changes in light scattering induced by folic acid are normal and cyclic AMP induces a monophasic instead of a biphasic response. The stimulatory effect of Con A on cyclic AMP mediated cyclic GMP accumulation is still observable at 10 ug Con A per ml. When cells are repeatedly stimulated with cyclic AMP, a decrease in light scattering without being accompanied by changes in cyclic GMP concentration is observed. Based on these results a model for chemotaxis is proposed. INTRODUCTION Vegetative cells of Dictyostelium discoideum are attracted by folic acid (Pan et al., 1972) and aggregative cells by cyclic AMP (Konijn et al., 1968). Both chemoattractants induce in cell suspensions of sensitive cells a fast increase of cyclic GMT?(Mato et al., 1977a and b; Wurster et al., 1977a) and a decrease of light scattering (Gerisch and Hess, 1974; Wurster and Schubiger, 1977). In D.lacteum and Polysphondilium violaceum cyclic GMP levels also increase in response to their specific attractants (Mato and Konijn, 1977; and Wurster et al., 1977b). In the present study we have investigated the role of concanavalin A (Con A) on attractant mediated cyclic GMP accumulation and light scattering decrease in cell suspensions of D.discoideum. Con A is known to increase the binding of cyclic AMP to the cellsurface of D.discoideum (Juliani and Klein, 1977) and to inhibit the chemotactic response to this attractant (Gillette and Filosa, 1973). Con A receptors have been visualized on the cell-surface of cells of D.discoideum (Molday et al., 1976) and a developmentally regulatedcarbohydrate binding protein, discoidin, has been purified from this species (Rosen et al., 1973).
164
Cell Biology
International
vegetative
aggregat
stage
stage
Reports,
Vol. 2, No. 2, 1978
ive
Control
Con A
++,,,-
100t.dml
Con
A
10 w/ml
7CAMP
f FA
/’
t FA
L *
TcAMP
Fig.1. Changes in light scattering in cell suspensions of D.discoideum in response to folic acid and cyclic AMP in the presence and absence of Con A. Cells were starved for 1 hour (vegetative stage) or for 5 hours (aggregative stage). When indicated by the arrow cells were stimulated with 3x10m6M folic acid (FA) or 3x10-k cyclic AMP.
Cell Biology International
Reports, Vol. 2, No. 2, 1978
165
RESULTS Effect of Con A on attractant induced decrease of light scattering. Incubation of cells of D.discoideum for 10 minutes with IO0 pg Con A per ml inhibits folic acid and cyclic AMP induced changes in light scattering completely (fig. 1). After 20 minutes acid inducincubation in the presence of 10 pg Con A per ml folic ed a normal decrease of light scattering (fig. 1). In contrast to the control, cells treated with 10 ug Con A per ml respond to cyclic AMP with a monophasic decrease in light scattering (fig. 1). After 10 minutes in the presence of 100 pg Con A per ml cells form fewer and smaller clumps than in the control, while in the presence of 10 ug Con A per ml the size and number of clumps is similar to that observed in the controls. In the presence of 1 mM EGTA cells also form very few and small clumps yet, they show a normal light scattering decrease in response to folic acid and cyclic AMP (fig. 2). This indicates that the inhibitory effect of Con A on light scattering is not due to differences in clump formation. The Con A ligand, a-D-glycopyranoside, counteracts the effect of Con A on light scattering (data not shown). Effect of Con A on attractant induced cyclic GMF' accumulation. In the presence of 100 ug Con A per ml vegetative cells of D.discoideum respond to folic acid with normal changes in the cellular content of cyclic GMP (fig. 3a). Contrary to what happens during the vegetative stage, aggregative cells treated with 100 ug Con A per ml respond to cyclic AMP with an increase in cyclic GMP double than that observed in the control (fig. 3b). The effect of Con A on cyclic AMP induced cyclic GMP accumulation is still observable at 10 pg Con A per ml (fig. 3b). Effect of cyclic AMP on light scattering and cyclic GMP when cells are triggered repeatedly. Fig. 4 shows the changes in light scattering and cyclic GNP when aggregative cells of D.discoideum are triggered repeatedly with cyclic AMP. A normal decrease in light scattering is induced by a second cyclic AMP pulse when given 30 seconds after a cyclic AMP pulse of the same magnitude. Contrary to what happens with light scattering, a second cyclic AMP pulse, only induces a very small, if any, change in the cyclic GMP content. DISCUSSION In D.discoideum incubation of cells for 5 minutes with 100 ug Con A per ml increases about twice the binding of cyclic AMF' to cell-surface receptors (Juliani and Klein, 1977). The present work indicates that the increased cyclic AMP binding in the presence of Con A is translated into an enhanced cyclic GMP accumulation in response to cyclic AMF'. Apparently, Con A unmasks functional receptors and did not induce unspecific binding. The Con A effect on atis developmentally regtractant mediated cyclic GMP accumulation ulated. This is shown by the lack of effect of Con A on folic acid
166
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
mediated cyclic GMP accumulation. A carbohydrate-binding protein has been purified from aggregative cells of D.discoideum (Rosen et al., 1973). The possibility that this protein activates attractant mediated cyclic GMP accumulation is under investigation. If so, this protein may not only be involved in cell to cell adhesion but also in the detection of a chemotactic signal. The inhibition by Con A of attractant induced changes in light scattering may also explain the decreased chemotaxis observed in D.discoideum in the presence of this lectin (Gillette and Filosa, 1973).
d
t FA
%4J t CAMP
Fig. 2. Changes in light scattering in cell suspensions of D.discoideum in the presence of 1 mM EGTA in response to folic acid and cyclic AMP. After starvation cells were washed twice in cold 10 mM phosphate buffer containing 1 mM EGTA, pH adjusted to 6.0, and resuspended in this same buffer at a cell density of 1x10* cells per ml. When indicated by the arrow vegetative cells (1 hour starvation) were triggered with 3x10e6M folic acid (FA) and aggregative cells (5 hours starvation) with 3x10-*M cyclic AMP.
Cell Biology
International
Reports,
167
Vol. 2, No. 2, 1978
We have previously shown (Mato et al., 1977a) that after triggering cells with 5x10m8M cyclic AMP a refractory period of 1 minute is induced. In contrast to these results, normal changes in light scattering are induced by a second cyclic AMP pulse when These results argue against a posgiven 30 set after the first. sible role of cyclic GMP in the generation of changes in light scattering. The results with Con A show that it is also possible to get changes in cyclic GMP without modifying the light scattering. Therefore, it is concluded that the interaction cyclic AMPa decrease in light scattering receptor(s) generates two signals: scattering and an increase in cyclic GMP. The decrease in light may be due to cell contraction (Wurster et al., 3977b). As shown is independent of by the experiments with EGTA, this contraction the presence of extracellular calcium (see also Gerisch et al., 1975).
7, a
14-
5,
10,
3-
6,
l-
21
0
L
20
I
I
40
60
1
Sec.
b
1
1
0
20
I
40
60
sec.
Fig. 3. Changes in cyclic GM? content in cell suspensions of D.discoideum in response to folic acid and cyclic AMP in the presence and absence of Con A.(a) Cells starven for I hour were stimulated at time zero with 3x10m6M folic acid: (0) control; and (0) preincubated for 10 minutes with 100 ug Con A per ml (b) Cells starven for 5 hours were stimulated at time zero with 3x10-* M cyclic AMP: (0) control; (4) preincubated for 20 minutes with 10 ng Con A per ml; and (0) preincubated for 10 minutes with 100 pg Con A per ml.
168
Cell Biology
International
Reports,
Vol. 2, No. 2, 7978
Cyclic GMPaccumulation in D.discoideum is also independent of extracellular calcium (Mato et al., 1977a) and in smooth muscle cyclic GMP accumulation has been correlated with cellular relaxation (Schultz et al., 1977). Based on these results we propose that chemotaxis in D.discoideum is the result of the following two processes: i) contraction over the whole cell, e.g. triggered by the release of calcium from intracellular deposits; and ii) formation of an intracellular gradient of cyclic GMPwhich causes the extension of a pseudopod by means of relaxation of the anterior part of the cell.
4
.
2 3 .I
0
-/
t
1
0
0.5
1
1.5
2
mi n.
T T Fig. 4. Effect of cyclic AMP on light scattering and cyclic GMP content when cells of D.discoideum are stimulated repeatedly. Cells starven for 5 hours were stimulated at time zero with 3x10-sM cyclic AMP and for a second time, with a pulse of the same magnitude, 30 set later.
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
169
MATERIALS AND METHODS D.discoideum, NC-4(H), was used for all experiments. Cells were grown and harvested as described by Konijn and Raper (1961). After centrifugation cells were resuspended in 10 mM phosphate buffer of lo7 cells per ml. Starvation was induced pH 6.0, at a density by shaking (Gerisch, 1962). After shaking, cells were centrifuged, washed twice in cold phosphate buffer, and adjusted to 1~10~ cells per ml. One portion of the suspension was then used for light scattering measurements and another portion for cyclic GMP determinations. Light scattering was recorded as described by Gerisch and Hess (1974) except that cells were aerated instead of oxygenated. For measuring the optical density at 405 nm a Beckman Acta III spectrophotometer was used. Cyclic GMP measurements were carried out as previously described (Mato et al., 1977 a and b). Material for the radioimmunoassay of cyclic GMP was provided by Amersham. Results are given as pmoles cyclic GMP per IO7 cells. Cyclic GMP and cyclic AMP were purchased from Sigma. Concanavalin A, was from Calbiochem.
from Boehringer. A, lyophilized
Folic acid was in NaCl, grade
ACKNOWLEDGMENTS The technical acknowledged.
assistance
of Miss
A. van Oosten
is gratefully
REFERENCES Gerisch, G. (1962) Zellfunktionen und Zellfunktionswechsel in der Entwicklung von Dictyostelium discoideum. IV. Der Zeitplan der Entwicklung. Wilhelm Roux Archiv fur Entwicklungsmechanik, -f153 603-520. oscillations Gerisch, G. and Hess, B. (1974) Cyc 1ic AMP controlled in suspended Dictyostelium cells: Their relation to morphogenetic cell interactions. Proceedings of the National Academy of Sciences USA, 71, 2118-2122. Gerisch, G., Malchow, D., Huesgen, A., Nanjundiah, V., ROOS, W. and Wick, U. (1975) Cyclic AMP reception and cell recognition in Dictyostelium discoideum. In: D.McMahon and C.F. Fox (eds.). Developmental Biology, ICN-UCLA symposia on Molecular and Cellular Biology. Vo1.2, pp.76-88. W.A. Benjamin, Inc. Gillette, M.U. and Filosa, M.F. (1973) Effect of Concanavalin A on cellular slime mold development: premature appearance of membrane-bound cyclic AMP phosphodiesterase. Biochemical and Bipphysical Research Communications, 53, 1159-1166. Juliani, M.H. and Klein, C. (1977) Calcium ion effects on cyclic adenosine 3':5' monophosphate bindings to the plasma membrane of Dictyostelium discoideum. Biochimica et Biophysics Acta, 497, 369-376.
170
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
in DictyosteKonijn, T.M. and Raper, K.B. (1961) Cell aggregation lium discoideum. Develop. Biol.,?, 725-756. Konijn, T.M., Barkley, D.S., Chang, Y.Y. and Bonner, J.T. (1968) occurring acrasin in the cellular slime Cyclic AMP: a naturally molds. American Naturalist, 102, 22.5-234. Mato, J.M., Krens, F.A., van Haastert, P.J.M. and Konijn, T.M. (1977a) Cyclic AMP dependent cyclic GMP accumulation in Dictyostelium discoideum. Proceedings of the National Academy of Sciences USA, 74, 2348-2351. P.J.M., Krens, F.A., Rhijnsburger, E.H., Mato, J.M., van Haastert, T.M. (1977b) Cyclic AMP and folic Dobbe, F.C.P.M. and Konijn, acid mediated cyclic GMP accumulation in Dictyostelium discoideum. FEBS Letters, 2, 331-336. Mato, J.M. and Konijn, T.M. (1977) The chemotactic signals and cyclic GMP accumulation in Dictyostelium. In: P. Cappuccinelli and J.M. Ashworth (eds.). Proceedings of the EMBO-workshop on Development and Differentiation in Cellular Slime Moulds. Vol.], pp.93-103. Elsevier, North-Holland. R. and McMahon, D. (1976) Concanavalin A and Molday, R., Jaffe, wheat germ agglutinin receptors on Dictyostelium discoideum. The Journal of Cell Biology, 71, 314-322. E.M. and Bonner, J.T. (1972) Folic acid as second Pan, P., Hall, chemotactic substance in the cellular slime moulds. Nature New Biology, 237, 181-182. Rosen, S.D., Kafka, J.A., Simpson, D.L. and Barondes, S.H. (1973) Developmentally regulated carbohydrate binding protein in Dictyostelium discoideum. Proceedings of the National Academy of Sciences USA, 70, 2554-2557. Schultz, K.D., Schultz, K. and Schultz, G. (1977) Sodium nitroprusside and other smooth muscle-relaxants increase cyclic GMP levels in rat ductus deferens. Nature, 265, 750-751. Wurster, B., Schubiger, K., Wick, U. and Gzsch, G. (1977a) Cyclic GMP in Dictyostelium discoideum: oscillations and pulses in response to folic acid and cyclic AMP signals. FEBS Letters, 76, 141-144. Wurster, B., Bozarro, S. and Gerisch, G. (1977b) Cyclic GMP regulation and responses of Polysphondylium violaceum to chemoattractants. Cell Biology, in press. Wurster, B. and Schubiger, K. (1977) Oscillations and cell development in Dictyostelium discoideum stimulated by folic acid pulses. Journal of Cell Science, in press.
