Abstracts of Communications 565th Meeting of the Biochemical Society University of Stirling 9 and 10 September 1976
CONTROL M ECHAN ISMS IN DIFFER ENTlAT1ON AND DEVELOPMENT: a Colloquium organized by L. Stevens (Stirling)
Control of Cell Differentiation in the Cellular Slime Mould Dictyostelium discoideum JOHN M . ASHWORTH Department of Biology, University of Essex, Colchester C04 3SQ, Essex, U.K. Cell differentiation is recognized and defined by the appearance, in a cell undergoing a differentiation process, of a novel chemical substance and/or novel biological activity. For the biochemist this implies that the cell must acquire novel enzymic activities during its differentiation, and much of the biochemical literature devoted to the problems posed by cell differentiation in fact consists of a more or less sophisticated analysis of the changes in enzymic activity, which accompany morphological changes in cells. In the specific case of the cellular slime mould Dictyostelium discoideum, the celldifferentiation process consists of the transformation of solitary amoeboid cells into either the spore or the stalk cells of the fruiting body, whose formation marks the end and the point of the cell-differentiation process. The most obvious novel chemicals produced during this process are the carbohydrates, namely cellulose (which occurs in both the stalk and the spore cell walls), a mucopolysaccharide (which occurs in the outermost layer of the spore wall and sticks the spore mass together) and trehalose (the characteristic storage product of the spore cell). Not surprisingly therefore much attention has been devoted to the enzymes that are involved in the biosynthesis of these materials and in particular to UDP-glucose pyrophosphorylase, trehalose 6-phosphate synthase and UDP-galactose-polysaccharide transferase. This work, which has been well summarized by Loomis (1975), has shown the following. (1) Characteristic changes in enzymic activity occur, which are correlated with specific morphological changes in the cell aggregates. (2) These changes in specific activity are inhibited by drugs that inhibit transcriptional and translational events. (3) In the specific case of UDP-glucose pyrophosphorylase, it has been shown that these increases in enzymic activity are paralleled by an increase in the cellular concentration of the enzyme (Franke & Sussman, 1973). It has thus been postulated that the developmental programme consists of a series of switches, which regulate the activity of the structural genes for enzymes whose activity
Vol. 4
33
BIOCHEMICAL SOCIETY TRANSACTIONS
962
is necessary for the developmentalprocess. Direct evidence for this hypothesis has come from the work of Dimond et al. (1976), who have isolated mutant strainsof D. discoideum, that are unable to synthesize UDP-glucose pyrophosphorylase. The amoebae of such strains grow normally and carry out the early stages of the developmental process, but are unable to form proper fruiting bodies and thus spore and stalk cells. This enzyme normally accumulatesduring the 12-20h period of developmentand the mutation manifests itself at the 18-20h period. It thus seems as if the enzyme plays no essential role during the time it is being accumulated. The key biochemical questions, that remain to be answered, thus concern the nature of the switches that regulate the synthesis and activity of these developmentally programmed enzymes, and particularly how these switchesare connected together to form a temporal sequence. Loomis et al. (1976) have pointed out that there are two general possibilities that must be considered.Either there is a dependent sequence (such as the cascade inductions occurring during the development of phage A), in which each stage depends on the attainment of previous stages or there is an independentsequencetriggered by the initial stimulus(Fig. 1). Analyses of the way in which morphologicalmutants do or do not synthesize various enzymic markers and, conversely,of the morphological consequencesof mutations in the structural genes for these enzymes suggest that the ‘Combination sequence’of Fig. 1 is the best description of the observed facts. What is postulated is a dependent sequence of events (A, B, C, D), which, together, define an invariant temporal sequence and a set of sequences(a, b, c, d), which are independent of one another, but are each dependent on the temporal sequence A, B, C, D (Loomis et al., 1976).. Turning now from the formal arrangement of the postulated switches to their biochemical nature we are again faced with two general possibilities. Either the switches are in some way dependent on cues from the metabolic events which they control or they
Independent sequence ( a ) Multiple timer -A
(6) Single timer
c
n
Dependent sequence A-B-C-D
- ---Combination Sequence
A
B
C
D
Fig. 1. Possible sequences of developmentallyprogrammed enzyme syntheses 1976
565th MEETING, STIRLING
963
are independent of such events and depend on cues they receive from the environment, perhaps mediated via the plasma and endoplasmic-reticulum membranes. Wright and her co-workers [whose work and views are well summarized in Wright (1973)l have argued vehemently for the first possibility and have even suggested that at some stages (particularly the earlier ones) the metabolic changes may occur before any necessary alterations in the cell's enzymic composition. In the particular case of UDP-glucose pyrophosphorylase it seems clear that the enzyme accumulates before the stage at which its activity becomes essential, but in other cases definitive evidence is not available. What is clear is that quite considerable stresses can be placed on the metabolic fluxes occurring during the differentiation process without necessarily causing any alterations in the cell's enzymic composition and, conversely, that alterations can be made in the cell's enzymic composition without causing any necessary dislocation of the cell-differentiation process. Experiments of the first type were described by Hames & Ashworth (1974), who showed that despite massive changes in the UDP-glucose and glucose 6-phosphate pools caused by preloading the differentiating cells with glycogen, there was no alteration in thecharacteristic changes that occurred in the specific activities of UDP-glucose pyrophosphorylase and trehalose 6-phosphate synthase, and this was despite the fact that such changes in pool size caused quite considerable (fourfold) changes in the amount of trehalose finally accumulated. It has, however, been possible to alter the amount of a developmentally regulated enzyme synthesized during the cell-differentiation process by altering the environmental conditions (Newell & Sussman, 1970), by the addition of the antibiotic formycin B (Brackenbury et al., 1974) or by mechanically disaggregating the cells and then allowing them to reassociate (Newell et al., 1971).
100 L,,-l0
~~~
16
~~
20
-
1
24
I
28
Time (h) Fig. 2. Synthesis of UDP-glucosepyrophosphorylase in diyerentiating cells on Millipore filters ( 0 ) and in cells dissociated enzymically at 18.5h and redeposited on Millipore filters (0) Dissociation and redeposition causes a 2 h increase in the time taken to form fruiting bodies, which are formed at 24h in the control cells (0).
Vol. 4
BIOCHEMICAL SOCIETY TRANSACTIONS
964
400
r
0
J
Total carbohydrate content of the cells w..Jse activity is recorded in Fig.2
Fig.
0 , Control
DP-glucose pyrophosphorj
cells; 0, dissociated cells.
We have used the enzymic disaggregation procedures of Takeuchi & Yabuno (1970) to repeat the experiments of Newell et al. (1971), and have shown that despite the increase in enzymic activity thereby induced (Fig. 2), there is in fact no parallel increase in the synthesis of end-product carbohydrates. Indeed, there can be (Fig. 3) a marked decrease in the amount of such carbohydrates synthesized. Thus two very different kinds of experiment lead to the same conclusion, namely that there is no necessary correlation between the amount of an enzyme synthesized during a developmental process and the use to which that enzymic activity will be put. Thus the kind of direct connexions between metabolic fluxes on the one hand and transcriptional controls on the other, which are so familiar to students of regulation in Escherichia coli, are unlikely to be of importance in controlling developmentally-programmed switches of the type discussed above. Instead we must look to specific ‘reporter molecules’ (such as cyclic AMP and guanosine tetraphosphate), which can by-pass the metabolic machinery in the cytoplasm and directly affect the nuclear regulatory mechanism. Brackenbury, R., Schindler, J., Alexander, S. & Sussman, M. (1974) J. Mol. Biol. 90, 529-539 Dimond, R., Farnsworth, P. A. & Loomis, W. F. (1976) Deuel. Biol. in the press Franke, J. & Sussman, M. (1973) J. Mol. Biol. 81, 173-185 Hames, B. D. & Ashworth, J. M. (1974) Biochem. J. 142,317-329 Loomis, W. F. (1975) Dictyostelium discoideum: A Developmental System, pp. 105-126, Academic Press, New York Loomis, W. F., Dimond, R., Free, S. & White, S. (1977) in Eukaryotic Microbes as Model Developmental Systems (ODay, D. & Horgan, P., eds.), Marcel Dekker, New York, in the press Newell, P. C. & Sussman, M. (1970) J. Mol. Biol. 49,627-637 Newell, P. C., Longlands, M. & Sussman, M. (1971) J. Mol. Biol. 58, 541-554 Takeuchi, I. & Yabuno, K. (1970) Exp. Cell Res. 61,183-190 Wright, B. E. (1973) Critical Variables in Diflerentiation, Prentice-Hall, New Jersey
1976
CONTROL M ECHAN ISMS IN DIFFER ENTlAT1ON AND DEVELOPMENT: a Colloquium organized by L. Stevens (Stirling)
Control of Cell Differentiation in the Cellular Slime Mould Dictyostelium discoideum JOHN M . ASHWORTH Department of Biology, University of Essex, Colchester C04 3SQ, Essex, U.K. Cell differentiation is recognized and defined by the appearance, in a cell undergoing a differentiation process, of a novel chemical substance and/or novel biological activity. For the biochemist this implies that the cell must acquire novel enzymic activities during its differentiation, and much of the biochemical literature devoted to the problems posed by cell differentiation in fact consists of a more or less sophisticated analysis of the changes in enzymic activity, which accompany morphological changes in cells. In the specific case of the cellular slime mould Dictyostelium discoideum, the celldifferentiation process consists of the transformation of solitary amoeboid cells into either the spore or the stalk cells of the fruiting body, whose formation marks the end and the point of the cell-differentiation process. The most obvious novel chemicals produced during this process are the carbohydrates, namely cellulose (which occurs in both the stalk and the spore cell walls), a mucopolysaccharide (which occurs in the outermost layer of the spore wall and sticks the spore mass together) and trehalose (the characteristic storage product of the spore cell). Not surprisingly therefore much attention has been devoted to the enzymes that are involved in the biosynthesis of these materials and in particular to UDP-glucose pyrophosphorylase, trehalose 6-phosphate synthase and UDP-galactose-polysaccharide transferase. This work, which has been well summarized by Loomis (1975), has shown the following. (1) Characteristic changes in enzymic activity occur, which are correlated with specific morphological changes in the cell aggregates. (2) These changes in specific activity are inhibited by drugs that inhibit transcriptional and translational events. (3) In the specific case of UDP-glucose pyrophosphorylase, it has been shown that these increases in enzymic activity are paralleled by an increase in the cellular concentration of the enzyme (Franke & Sussman, 1973). It has thus been postulated that the developmental programme consists of a series of switches, which regulate the activity of the structural genes for enzymes whose activity
Vol. 4
33
BIOCHEMICAL SOCIETY TRANSACTIONS
962
is necessary for the developmentalprocess. Direct evidence for this hypothesis has come from the work of Dimond et al. (1976), who have isolated mutant strainsof D. discoideum, that are unable to synthesize UDP-glucose pyrophosphorylase. The amoebae of such strains grow normally and carry out the early stages of the developmental process, but are unable to form proper fruiting bodies and thus spore and stalk cells. This enzyme normally accumulatesduring the 12-20h period of developmentand the mutation manifests itself at the 18-20h period. It thus seems as if the enzyme plays no essential role during the time it is being accumulated. The key biochemical questions, that remain to be answered, thus concern the nature of the switches that regulate the synthesis and activity of these developmentally programmed enzymes, and particularly how these switchesare connected together to form a temporal sequence. Loomis et al. (1976) have pointed out that there are two general possibilities that must be considered.Either there is a dependent sequence (such as the cascade inductions occurring during the development of phage A), in which each stage depends on the attainment of previous stages or there is an independentsequencetriggered by the initial stimulus(Fig. 1). Analyses of the way in which morphologicalmutants do or do not synthesize various enzymic markers and, conversely,of the morphological consequencesof mutations in the structural genes for these enzymes suggest that the ‘Combination sequence’of Fig. 1 is the best description of the observed facts. What is postulated is a dependent sequence of events (A, B, C, D), which, together, define an invariant temporal sequence and a set of sequences(a, b, c, d), which are independent of one another, but are each dependent on the temporal sequence A, B, C, D (Loomis et al., 1976).. Turning now from the formal arrangement of the postulated switches to their biochemical nature we are again faced with two general possibilities. Either the switches are in some way dependent on cues from the metabolic events which they control or they
Independent sequence ( a ) Multiple timer -A
(6) Single timer
c
n
Dependent sequence A-B-C-D
- ---Combination Sequence
A
B
C
D
Fig. 1. Possible sequences of developmentallyprogrammed enzyme syntheses 1976
565th MEETING, STIRLING
963
are independent of such events and depend on cues they receive from the environment, perhaps mediated via the plasma and endoplasmic-reticulum membranes. Wright and her co-workers [whose work and views are well summarized in Wright (1973)l have argued vehemently for the first possibility and have even suggested that at some stages (particularly the earlier ones) the metabolic changes may occur before any necessary alterations in the cell's enzymic composition. In the particular case of UDP-glucose pyrophosphorylase it seems clear that the enzyme accumulates before the stage at which its activity becomes essential, but in other cases definitive evidence is not available. What is clear is that quite considerable stresses can be placed on the metabolic fluxes occurring during the differentiation process without necessarily causing any alterations in the cell's enzymic composition and, conversely, that alterations can be made in the cell's enzymic composition without causing any necessary dislocation of the cell-differentiation process. Experiments of the first type were described by Hames & Ashworth (1974), who showed that despite massive changes in the UDP-glucose and glucose 6-phosphate pools caused by preloading the differentiating cells with glycogen, there was no alteration in thecharacteristic changes that occurred in the specific activities of UDP-glucose pyrophosphorylase and trehalose 6-phosphate synthase, and this was despite the fact that such changes in pool size caused quite considerable (fourfold) changes in the amount of trehalose finally accumulated. It has, however, been possible to alter the amount of a developmentally regulated enzyme synthesized during the cell-differentiation process by altering the environmental conditions (Newell & Sussman, 1970), by the addition of the antibiotic formycin B (Brackenbury et al., 1974) or by mechanically disaggregating the cells and then allowing them to reassociate (Newell et al., 1971).
100 L,,-l0
~~~
16
~~
20
-
1
24
I
28
Time (h) Fig. 2. Synthesis of UDP-glucosepyrophosphorylase in diyerentiating cells on Millipore filters ( 0 ) and in cells dissociated enzymically at 18.5h and redeposited on Millipore filters (0) Dissociation and redeposition causes a 2 h increase in the time taken to form fruiting bodies, which are formed at 24h in the control cells (0).
Vol. 4
BIOCHEMICAL SOCIETY TRANSACTIONS
964
400
r
0
J
Total carbohydrate content of the cells w..Jse activity is recorded in Fig.2
Fig.
0 , Control
DP-glucose pyrophosphorj
cells; 0, dissociated cells.
We have used the enzymic disaggregation procedures of Takeuchi & Yabuno (1970) to repeat the experiments of Newell et al. (1971), and have shown that despite the increase in enzymic activity thereby induced (Fig. 2), there is in fact no parallel increase in the synthesis of end-product carbohydrates. Indeed, there can be (Fig. 3) a marked decrease in the amount of such carbohydrates synthesized. Thus two very different kinds of experiment lead to the same conclusion, namely that there is no necessary correlation between the amount of an enzyme synthesized during a developmental process and the use to which that enzymic activity will be put. Thus the kind of direct connexions between metabolic fluxes on the one hand and transcriptional controls on the other, which are so familiar to students of regulation in Escherichia coli, are unlikely to be of importance in controlling developmentally-programmed switches of the type discussed above. Instead we must look to specific ‘reporter molecules’ (such as cyclic AMP and guanosine tetraphosphate), which can by-pass the metabolic machinery in the cytoplasm and directly affect the nuclear regulatory mechanism. Brackenbury, R., Schindler, J., Alexander, S. & Sussman, M. (1974) J. Mol. Biol. 90, 529-539 Dimond, R., Farnsworth, P. A. & Loomis, W. F. (1976) Deuel. Biol. in the press Franke, J. & Sussman, M. (1973) J. Mol. Biol. 81, 173-185 Hames, B. D. & Ashworth, J. M. (1974) Biochem. J. 142,317-329 Loomis, W. F. (1975) Dictyostelium discoideum: A Developmental System, pp. 105-126, Academic Press, New York Loomis, W. F., Dimond, R., Free, S. & White, S. (1977) in Eukaryotic Microbes as Model Developmental Systems (ODay, D. & Horgan, P., eds.), Marcel Dekker, New York, in the press Newell, P. C. & Sussman, M. (1970) J. Mol. Biol. 49,627-637 Newell, P. C., Longlands, M. & Sussman, M. (1971) J. Mol. Biol. 58, 541-554 Takeuchi, I. & Yabuno, K. (1970) Exp. Cell Res. 61,183-190 Wright, B. E. (1973) Critical Variables in Diflerentiation, Prentice-Hall, New Jersey
1976
