DEVELOPMENTAL
Synthesis
BIOLOGY
60,
207-216 (1977)
of Developmentally
Regulated Proteins in Dictyostelium on Continued Cell-Cell Interaction
discoideum Which Are Dependent THOMAS Department
H. ALTON’
of Biology, Massachusetts
AND
Institute
HARVEY
of Technology,
Received March 10,1977;
F. LODISH~ Cambridge,
Massachusetts
02139
accepted June 9,1977
In the previous paper we showed that the major changes in the pattern of protein synthesis during differentiation of Dictyostelium discoideum occur during the 4-hr period when the cells are forming tight, visible aggregates. During this time, synthesis of 10 discrete polypeptides made by preaggregation cells ceases or is reduced considerably, and synthesis of 40 new proteins is induced. Induction or cessation of synthesis of these proteins was parallelled by the appearance or disappearance of the corresponding messenger RNAs. In this paper we show that many of these changes are induced by continued cell-cell contact. None of these occurs in aggregation-competent cells kept in suspension culture, but changes do take place when such cells are allowed to form tight aggregates. Disaggregation of cells causes cessation of synthesis of “aggregation-stage” proteins and reinduction of synthesis of polypeptides characteristic of preaggregation cells. INTRODUCTION
We have been investigating the changes in the pattern of protein synthesis during development of the cellular slime mold Dictyostelium discoideum (Alton and Lodish, 1977). The major changes occur between 8 and 10 hr, coincident with the formation of tight aggregates. During this time, synthesis of 10 proteins is reduced considerably, and 40 proteins begin being synthesized or show greatly enhanced relative synthesis. This observation led us to hypothesize that the synthesis of certain proteins is regulated by some event associated with cell-cell aggregation. In this paper we provide demonstration that indeed many of these changes in the pattern of protein synthesis are induced by continued cell-cell interactions in these tight aggregates. The notion that there is a relationship between morphology and gene expression ’ Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305. * To whom reprint requests should be sent.
in Dictyostelium is not new. Newell et al. (1971, 1972) demonstrated that physical perturbation of normal development resulted in altered patterns of accumulation of several enzymatic activities. The essence of their results is that certain enzymes accumulate only when cells form tight aggregates and that continued accumulation of these enzymes depends on continued morphological integrity. Furthermore, the kinetics of accumulation of certain enzymes is dependent upon whether the aggregates form migrating slugs or proceed directly to culmination. Development in Dictyostelium is initiated by removing nutritional sources, specifically any of several amino acids (Marin, 1976). Approximately 3-4 hr after the outset of starvation, the cells become responsive to pulses of CAMP and capable of relaying the pulses (Robertson and Cohen, 1972). By 8 hr, the cells are cohesive, possess surface antigens not found in growing cells (Beug et al., 19731, and contain high levels of a carbohydrate-binding protein (Rosen et al., 1973). These changes 207
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0012-1606
208
DEVELOPMENTAL BIOLOGY
take place whether the cells are plated onto a solid surface or are kept shaking in suspension. Thus, starvation leads to the acquisition of aggregation competence (Gerisch, 1968). Once the cells have becompetent, further come aggregation changes in CAMP receptor and phosphodiesterase levels (Henderson, 1975) require that the cells be allowed to form tight aggregates. In this paper we have tested the hypothesis that synthesis of the “aggregationstage” proteins by Dictyostelium requires continuous cell contact. By studying the effects of disaggregation and reaggregation on protein synthesis, we conclude that continued cell contact is required for synthesis of a number of proteins and, presumably, for induction of transcription of the genes encoding these mRNAs. Continued cell contact is also required for cessation of synthesis of several proteins whose synthesis is characteristic of preaggregation cells. MATERIALS
AND
METHODS
All procedures for cell culture, differentiation, labeling with [35S]methionine, and analysis of radioactive proteins by electrophoresis on two-dimensional polyacrylamide gels (C)‘Farrell, 1975) are detailed in the previous paper (Alton and Lodish, 1977). In all cases cells were labeled for 15 min. Also in this paper is a discussion of the quantitation and limitations of this gel technique. Suspension cells are prepared by harvesting vegetative amoebas and resuspending them at 5 x lo6 cells/ml in MES-PDF, the normal buffer used for plating cells. These starved cultures were aerated by shaking at 22°C and at 150 rpm. Even after 20 hr, aggregates consisting of only a few cells could be seen. Disaggregated cells were prepared by washing cells from the filter pads into plating buffer. The cells were diluted to 5 x lo6 cells/ml and separated into single cells by rejected vigorous pipetting. These manipulations required less than 2 min.
VOLUME 60, 1977 RESULTS
Changes in the pattern of protein synthesis during development of DictyosteZium are discussed in detail in the previous paper (Alton and Lodish, 1977). For clarity, relevant features are reviewed here. Cells at the appropriate stage were labeled with [35S]methionine for 15 min. At the end of the label period, samples were prepared for 2-D gel electrophoresis. Labeled polypeptides were assayed by a modification of the two-dimensional gel system devised by O’Farrell (1975). The first dimension consists of isoelectric focusing in the presence of 9 M urea. The second dimension is electrophoresis in polyacrylamide gels containing SDS and an exponential gradient of polyacrylamide (Alton and Lodish, 1977). Between 4 and 10 hr, numerous changes in the pattern of protein synthesis can be detected by 2-D gel electrophoresis (Figs. 1 and 2). Nearly all of these changes occur after the cells have aggregated into discrete tight mounds (8 hr). Many new polypeptide species are synthesized by lo-hr cells which are not synthesized by cells before 7 hr (spots 43, 44, 46-50, 52-55, 57, 60, 62-69). Several other polypeptides show increased relative rates of synthesis (spots 19, 33, 45, 51, 56, 59, 61, 84). Simultaneously, several proteins show reduced rates of synthesis (spots 2-5, 13, 16, 23, 25, 31, 58) or cease being synthesized altogether (spot 70). Many other proteins show no detectable changes in the relative rate of synthesis. In order to test whether these changes in the patterns of protein synthesis and translatable mRNAs were affected by the formation of tight cell contacts which occur at 10 hr, we studied the pattern of proteins made by aggregation-competent cells. These are cells which were removed from growth medium and resuspended at 5 x lo6 cells/ml in the normal buffer used for cell plating (MES-PDF) but which were shaken at 110 rpm at 22°C. At this low cell density, shaking completely prevents the
ALTON AND LODISH
Developmentally
Regulated
Protein
Synthesis
209
FIG. 1. Two-dimensional gel of proteins synthesized by plated cells 4 hr after initiation of differentiation. Six million Dictyostelium cells were labeled with 50 $i of [35Slmethionine from 4 to 4.25 hr after initiation of differentiation. The cells and extracellular fluid were harvested, combined, and prepared for gel electrophoresis by boiling in a solution containing sodium dodecyl sulfate. The first dimension on these gels is isoelectric focusing from pH 3 (left) to pH 10 (right); the second dimension is electrophoresis in a gradient of polyacrylamide gel containing SDS. Larger proteins migrate closer to the top of the gel. The molecular weight of actin (43,000) serves as a calibration for the sizes of other proteins. The fluorogram of the gel was exposed to the equivalent of log cpm added for 24 hr. All of the details concerning the gel procedure are given in the previous paper (Alton and Lodish, 1977).
formation of cell-cell contacts. If cells are starved in suspension from between 5 and 20 hr and then plated for development, they will form aggregates in 3 hr, rather than the 8 hr normally required for differentiating cells. Thus, starved cells kept in suspension are said to be “aggregation competent” (Gerisch, 1968). A comparison of Figs. 1 and 3 shows that the pattern of protein synthesized by cells starved for 5 hr in suspension is very similar to that of cells plated for normal differentiation for 6 (or 3 or 8) hr. Synthesis continues of polypeptides characteristic of 4-hr differentiating cells (Nos. 3, 13, 21, 23, 58, and 70 at high levels; and 19, 33, 45, 51, 59, and 81 at low levels). Figure 4 shows the pattern of protein synthesis of cells starved in suspension for 20 hr. The pattern is very similar to that of
cells starved in suspension for 5 hr, although there are quantitative differences in the relative rates of synthesis of certain polypeptides. Important is the fact that these cells continue synthesis of several polypeptides (Nos. 13, 21, 30, 31, 45, 51, 58, and 70) whose synthesis is characteristic of preaggregation cells. Also of significance is the result that these aggregation-competent cells do not synthesize any of the polypeptides whose synthesis is characteristic of lo-hr postaggregation cells (for instance, polypeptides 43, 44, 46, 47, 48, 49, 50, 52-55, 57, 60, 62-69). It is concluded that cells, starved in suspension and competent to aggregate, do not exhibit most of the changes in the pattern of protein synthesis characteristic of normal late-aggregation-stage cells. They continue to synthesize a pattern of proteins characteristic
210
DEVEKIPMENTAL
FIG. 2. Two-dimensional gel of proteins cells were labeled 10 hr after plating.
BIOLOGY
synthesized
VOLUME
60, 1977
by cells plated for 10 hr. Same as Fig. 1, except that
FIG. 3. Two-dimensional gel of proteins synthesized by cells in suspension for 5 hr. Same as Fig. 1, except that cells were labeled after starvation for 5 hr in suspension.
of interphase or early-aggregation-stage cells. Thus suspension cells, mechanically prevented from aggregating, appear to
proceed more or less normally through the first 6 to 8 hr of differentiation, but then remain “locked” into a pattern of protein
ALTON
AND
LODISH
Developmentally
Regulated
Protein
FIG. 4. Two-dimensional gel of proteins synthesized by cells in suspension except that cells were labeled after starvation for 20 hr in suspension.
synthesis characteristic of that stage. They exhibit none of the changes which normally occur during or after late aggregation. When cells which have been starved in suspension for 5-20 hr are plated on filter pads, they aggregate within 3 hr. Concomitantly, they cease synthesis of polypeptide 70 and reduce synthesis of 13, 16, 25, 31, and 58, whose production is characteristic of normal preaggregation cells. They also begin synthesis of many (but not all) of the proteins whose synthesis is characteristic of normal lo-hr differentiating cells: peptides 43, 44, 47, 50, 54, 55, 57, 60, 63-69 (Fig. 5, Table 1). In the case of those proteins whose mRNAs could be identified by translation in a cell-free system, we showed that synthesis of these aggregation-stage polypeptides is accompanied by appearance of the homologous translatable mRNA. We conclude that the formation of tight cell-cell contacts is necessary for the induction of synthesis of these proteins and, presumably, for initiation of transcription of the homologous DNA genes.
Synthesis
211
for 20 hr. Same as Fig. 1,
Disaggregation The experiments in Fig. 6 and Table 1 show that continued cell-cell contact is essential for continued synthesis of these characteristic aggregation stage proteins. When cells are plated normally for 5 hr, then removed from the filter pads and placed in suspension at low density for 5 hr, they fail to synthesize many of the proteins which are made by normal lo-hr cells (Fig. 6). They continue synthesizing polypeptides such as 70 whose synthesis is characteristic of preaggregation cells. Even when cells which have been plated for 10 hr and which have formed tight cellcell aggregates are removed from the filter and shaken hard in suspension to disaggregate clumps of cells, they cease synthesis of many of the characteristic lo-hr aggregation-stage proteins and begin or increase synthesis of proteins characteristic of early aggregates (spots 13, 58,701 (Table 1). Polypeptides such as 43, 44, 54, 57, 66, 67, 68, and 69 are made by normal lo-hr cells in tight aggregates but not when these cells have been placed into suspen-
212
DEVEWPMENTAL
BIOLOGY
VOLUME 60, 1977
FIG. 5. Two-dimensional gel of proteins synthesized by cells 5 hr after plating preceded by 5 hr in suspension. Cells harvested from growth medium were kept in suspension for 5 hr before plating. After 5 hr on plates, the cells were labeled for 15 min as described in Fig. 1. At this time the cells had formed aggregates morphologically similar to normal lo-hr differentiating cells. TABLE
1
PROTEIN SYNTHESIS IN PLATED CELLS, SUSPENSION CELLS, DISAGGREGATED CELLS AND REAGGREGATED CELL@ Protein
13 16 33 43 44 45 48 49 50 51 54 57 58 60 65 66 67 68 69 70
Plated
Suspension/ plated
Suspension
Disaggregatedl reaggregated
5
10
5
5
10
15
20
515
1015
515
IO/5
++ ++ ” 0 0 2 0 0 0 + 0 0 + 0 0 0 0 0 0 +
” + + + + + + + + + + + k + + + + + + 0
? + + + + + + + + + + + 2 + + + + + + 0
+ + k 0 0 t 0 0 0 0 0 0 + 0 0 0 0 0 0 +
+ + f 0 0 k 0 0 0 0 0 0 + 0 0 0 0 0 0 +
+ + _t 0 0 i0 0 0 0 0 0 + 0 0 0 0 0 0 +
+ + 2 0 0 + 0 0 0 0 0 0 + 0 0 0 0 0 0 +
+ + + + + + 0 + + + + + 2 + + + + + ++ 0
t 0 + + 0 + 0 0 0 + 0 + k 0 0 + + + ++ 0
L 5 + + + + -t + + 5 + + 5 + + + + + -t 0
+ k + + 0 + 0 + 0 0 + ++ t 0 + + 0 + k 0
Disaggregatedl suspension 515
+ ++ + 0 0 k 0 0 0 f 0 0 + 0 0 0 0 0 0 +
1015
+ + k 0 0 f 0 0 0 -t 0 + + 0 f ” 0 0 t +
a The numbers at the head of each column indicate the time in hours since removal of the cells from growth medium. Numbers such as 5/5 indicate that cells were either on plates or in suspension for 5 hr, then shifted to another condition for 5 hr before labeling of the cells. The 0, +, + , and + + indicate the relative rate of synthesis.
ALTON
AND LODISH
Developmentally
Regulated
Protein
Synthesis
213
FIG. 6. Two-dimensional gel of proteins synthesized by cells plated for 5 hr, disaggregated, and kept in suspension for 5 hr. Cells were allowed to differentiate normally on plates for 5 hr, then were disaggregated and starved in suspension culture for an additional 5 hr. At this point they were labeled for 15 min with [%lmethionine. Gel analysis is as in Fig. 1.
sion culture. Low levels of polypeptides 57, 66, and 69 are occasionally made by cells which have been plated for 10 hr and then kept in suspension for 5. Presumably this occurs because some mRNA encoding these proteins persists after the removal to suspension culture, but that additional mRNA encoding these proteins is not made after removal from the plates. Synthesis of protein 70 is characteristic of preaggregation but not of lo-hr plated cells. It is of interest that synthesis of this “preaggregation” protein resumes when lo-hr cells are placed in suspension (Table 1). Presumably this is due to resynthesis of mRNA encoding this protein since translatable mRNA for protein 70 is not present (or is present in low levels) in normal cells at 10 hr of differentiation (Alton and Lodish, 1977). Manipulations associated with disaggregation or reaggregation are not, in themselves, sufficient to affect the pattern of protein synthesis. If cells at 10 or 15 hr of normal differentiation are removed from
the filter, disaggregated, and then replated, they will form tight aggregates (characteristic of normal lo-hr differentiating cells) within 3 hr. The pattern of proteins made by these disaggregated and replated cells is that characteristic of normal cells (Table 1). There are some stage-specific proteins which do not fall into categories such as “aggregation induced” or “aggregation repressed.” Some proteins which are synthesized by cells at 10 hr of normal (plated) development are not synthesized by cells which are removed from filters at 5 hr after plating, dissociated, and then replated for 5 hr before labeling (spots 44,48, 51, and 601, in spite of the fact that these cells form morphologically normal aggregates. Some proteins are synthesized by aggregated cells which were plated immediately upon removal from growth medium and allowed to develop for 10 or 15 hr, but are not synthesized by aggregated cells which were kept in suspension for 5 or 10 hr and then plated for 5 hr before
214
DEVELOPMENTAL BIOLOGY
labeling (spots 48 and 49). Furthermore, there are proteins which are synthesized by aggregating cells incubated for 5 hr in suspension followed by 5 hr on filters, but which are not synthesized by cells kept 10 hr in suspension followed by 5 hr on filters (peptides 44, 50, 52, 60, and 65). DISCUSSION
The principal conclusion of this work is that continued cell-cell contact is essential for synthesis of a number of developmentally regulated proteins which are characteristic of postaggregation cells. We that translatable showed previously mRNAs encoding these proteins can be detected only in postaggregation cells (Alton and Lodish, 1977). Thus continued cell contact is required for the formation of translatable mRNAs for these characteristic proteins and, presumably, for transcription of the appropriate genes. While there is no evidence for “masked” cytoplasmic mRNA species (Alton and Lodish, 1977), and there is little processing involved in formation of Dictyostelium mRNA (Lodish et al., 1973; Dottin et al., 1976; Weiner and Lodish, in preparation), we cannot eliminate the possibility that appearance of these translatable mRNAs is regulated at a post-transcriptional level. Cell Contact During
Late Aggregation
The changes in the pattern of protein synthesis which occur during the first 8 hr of normal differentiation (interphase and early aggregation) also occur in cells which have been starved in suspension instead of being plated. A few differences in the pattern of protein synthesis can be detected between plated and suspension cells, but these differences most likely reflect physiological rather than developmental differences. Cells starved in suspension show very few changes after the first 5 hr. The pattern of protein synthesis in cells kept in suspension for 20 hr is virtually indistinguishable from the pattern of protein syn-
VOLUME 80, 1977
thesis of cells in suspension for 5 hr (Figs. 2-4). These suspension cells are competent to aggregate and will do so within 2 hr after plating. They contain levels of CAMP phosphodiesterase, CAMP surface receptors, and several other surface molecules characteristic of normal aggregating cells (Beug et al., 1973; Henderson, 1975; Huesgen and Gerisch, 1975). But acquisition of the state of “aggregation competence” is insufficient to induce synthesis of mRNAs and proteins which are characteristic of aggregating cells. Cells which are plated exhibit many changes in the pattern of protein synthesis during late aggregation. During this time the cells form tight mounds of approximately lo5 cells each. Cells which have been kept in suspension and then plated show many of these same changes when the morphology of these aggregates is identical to that of a normal late aggregate. Furthermore, the changes can be prevented or even reversed by removing cells from the filter papers, dispersing the aggregates into single cells, and keeping them in suspension for 5 hr. These data lead to the hypothesis that cell contact plays an essential role in the normal developmental program. Cell contact acts by inducing transcription of new genes and repressing transcription of other genes. Furthermore, continued cell contact is essential for continued expression of the genes activated by cell contact, and for continued repression of the genes repressed by cell contact. Further insight into this problem has come from a study of mutants which are unable to aggregate (Lodish et al., 1976; Margolskee and Lodish, in preparation). The pattern of protein synthesis by two independent mutants which show no signs of aggregation was studied by two-dimensional gel electrophoresis. All such mutants show the same pattern of protein synthesis as do wild-type cells for the first 6 hr after plating. However, these mutants continue making most of the “early” pro-
ALTON
AND LODISH
Developmentally Regulated Protein Synthesis
teins, including actin, for at least 24 hr and exhibit no induction of polypeptides synthesized in wild-type cells during aggregation. Although we do not know the primary defect in any of these mutants, this work is consistent with our conclusion that cell-cell contact is required for induction of most aggregation-stage proteins. Previous work suggested that cell contact might affect expression of certain developmentally regulated genes, but the experiments were less direct than those detailed here. Mutants which are unable to aggregate generally do not accumulate the levels of activities of certain enzymes which are characteristic of aggregating cells (Loomis et al., 1976). The necessity of continuous cell contact for maintenance of the late aggregationpostaggregation state was demonstrated by Okamoto and Takeuchi (1976). Postaggregation-stage cells were disaggregated and resuspended as single cells, and the activity of two enzymes was monitored. The activity of cell-bound CAMP-bound phosphodiesterase, an enzyme characteristic of preaggregating cells, began to increase immediately, and the activity of UDP-galactose polysaccharide transferase, an activity characteristic of late differentiating cells, began to decrease immediately. Thus these disaggregated cells came to resemble cells which had not aggregated. Newell et al. (1971,1972) showed that continued cell aggregation was essential for appearance of enzymes specific for later morphological stages. The effects of cell contact on induction of new gene functions described here differ from the cases of embryonic induction of new gene functions. Embryonic induction requires generally the interaction of two different tissues, and one tissue is induced to differentiate by contact with the other. the changes in the patIn Dictyostelium tern of protein synthesis (and therefore in the pattern of gene expression) do not require the interaction of two different tissues, but rather the presence of a number
215
of cells in contact with each other. The “induction phenomenon” in slime molds is a self-induction rather than a heterologous induction. However, the mechanism of induction may well be similar in slime molds and in organogenesis in higher organisms. Smart and Tuchman (1976) demonstrated that autoclaved membranes isolated from late-aggregation- or early-postaggregation-stage cells inhibit aggregation of cells plated in the presence of these membrane preparations. Reaggregation of cells that had formed tight aggregates and then disaggregated was not inhibited by these membranes. Tuchman et al. (1976) investigated the effects of these membrane preparations on the accumulation of several developmentally regulated enzymes. The accumulation of one early enzyme, threonine deaminase, was essentially identical in membrane-treated and untreated cells, but the normal decrease in activity characteristic of later development did not occur in the treated cells. Accumulation of another early enzyme, N-acetylglucosaminidase, was completely inhibited in membrane-treated cells. Accumulation of UDPglucose-pyrophosphorylase and tyrosine transaminase (both late-aggregation enzymes) and P-glucosidase 2 (a culmination-specific enzyme) was also blocked. Thus membrane preparations from lateaggregation cells do have effects on the developmental program, and probably act primarily by preventing formation of normal cell-cell contacts. The elucidation of the factor (or factors) responsible for the cell contact effects awaits further investigation. It is not clear from our work whether direct cell contact is required or whether the concentration of a diffusible factor produced by aggregating cells must be above a critical level in order to induce the changes in the pattern of protein synthesis. This research was supported by Grant PCM7404869 A02 from the National Science Foundation. We thank Naomi Cohen and Martin Brock for ex-
216
DEVEU)PMENTAL BIOLOGY
pert technical assistance and Marianne Robotham for going blind and crazy over deciphering the original draft. REFERENCES ALTON, T., and LODISH, H. F. (1977). Developmental changes in messenger RNAs and protein synthesis in Dicytostelium discoideum. Develop. Biol. 180-206. BEUG, H., KATZ, F. E., and GERISCH, G. (1973). Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum. J. Cell Biol. 56, 647-658. DOTTIN, R. P., WEINER, A. M., and LODISH, H. F. (1976). 5’ Terminal nucleotide sequences of the messenger RNAs of Dictyostelium discoideum. Cell 8, 233-244. GERISCH, G. (1968). Cell aggregation and differentiation in Dictyostelium. In “Current Topics in Developmental Biology” (A. A. Moscona and A. Monroy, eds.), Vol. 3, pp. 159-197. Academic Press, New York. HENDERSON, E. J. (1975). The cyclic adenosine 3’:5’monophosphate receptor of Dictyostelium discoi&urn. Binding characteristics of aggregation competent cells and variation of binding levels during the life cycle. J. Biol. Chem. 250, 4730-4736. HEUSCEN A., and GERISCH, G. (1975). Solubilized contact sites A from cell membranes of Dictyostelium discoideum. FEBS Lett. 56, 46-49. LODISH, H. F., FIRTEL, R. A., and JACOBSON, A. (1973). Transcription and structure of the genome of the cellular slime mold Dictyostelium discoideum. Cold Spring Harbor Symp. 38, 899-914. LODISH, H. F., ALTON, T., DOTTIN, R. P., WEINER, A. M., and MARGOL~KEE, J. P. (19761. Synthesis and translation of messenger RNA during differentiation of the cellular slime mold Dictyostelium discoideum. In “The Molecular Biology of Hormone Action” (J. Papaconstantinou, ed.), pp. 75-106. Academic Press, New York. LOOMIS, W. F., WHITE, S., and DIMOND, R. L. (1976).
VOLUME 60, 1977
A sequence of dependent stages in development of Dictyostelium discoideum. Develop. Biol. 53, 171177. MARIN, F. T. (1976). Regulation of development in Dictyostelium discoideum. I. Initiation of the growth to development transition by amino acid starvation. Develop. Biol. 48, 110-117. NEWELL, P. C., LONGLANDS, M., and SUSSMAN, M. (1971). Control of enzyme synthesis by cellular interaction during development of the cellular slime mold Dictyostelium discoideum. J. Mol. Biol. 58, 541-554. NEWELL, P. C., FRANKE, J., and SUSSMAN, M. (1972). Regulation of four functionally related enzymes during shifts in the developmental program ofDictyostelium discoideum. J. Mol. Biol. 63, 373382. O’FARRELL, P. H. (1975). High resolution two-dimensional gel analysis of proteins. J. Biol. Chem. 250, 4007-4021. OKAMOTO, K., and TAKEUCHI, I. (1976). Changes in activities of two developmentally regulated enzymes induced by lisaggregation of the pseudoplasmodia of Dictyostelium discoideum. Biochem. Biophys. Res. Commun. 72, 739-746. ROBERTSON, A., and COHEN, M. H. (1972). Control of developing fields. Annu. Rev. Biophys. Bioengineer. 1, 409-464. ROSEN, S. D., KAFKA, J. A., SIMPSON, D. L., and BARONDES, S. H. (1973). Developmentally regulated, carbohydrate-binding protein in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 70, 2554-2557. SMART, J. E., and TUCHMAN, J. (1976). Inhibition of the development of Dictyostelium discoideum by isolated plasma membranes. Develop. Biol51, 6376. TUCHMAN, J., SMART, J. E., and LODISH, H. F. (1976). Effects of differentiated membranes on the developmental program of the cellular slime mold. Develop. Biol. 51, 77-85.
Synthesis
BIOLOGY
60,
207-216 (1977)
of Developmentally
Regulated Proteins in Dictyostelium on Continued Cell-Cell Interaction
discoideum Which Are Dependent THOMAS Department
H. ALTON’
of Biology, Massachusetts
AND
Institute
HARVEY
of Technology,
Received March 10,1977;
F. LODISH~ Cambridge,
Massachusetts
02139
accepted June 9,1977
In the previous paper we showed that the major changes in the pattern of protein synthesis during differentiation of Dictyostelium discoideum occur during the 4-hr period when the cells are forming tight, visible aggregates. During this time, synthesis of 10 discrete polypeptides made by preaggregation cells ceases or is reduced considerably, and synthesis of 40 new proteins is induced. Induction or cessation of synthesis of these proteins was parallelled by the appearance or disappearance of the corresponding messenger RNAs. In this paper we show that many of these changes are induced by continued cell-cell contact. None of these occurs in aggregation-competent cells kept in suspension culture, but changes do take place when such cells are allowed to form tight aggregates. Disaggregation of cells causes cessation of synthesis of “aggregation-stage” proteins and reinduction of synthesis of polypeptides characteristic of preaggregation cells. INTRODUCTION
We have been investigating the changes in the pattern of protein synthesis during development of the cellular slime mold Dictyostelium discoideum (Alton and Lodish, 1977). The major changes occur between 8 and 10 hr, coincident with the formation of tight aggregates. During this time, synthesis of 10 proteins is reduced considerably, and 40 proteins begin being synthesized or show greatly enhanced relative synthesis. This observation led us to hypothesize that the synthesis of certain proteins is regulated by some event associated with cell-cell aggregation. In this paper we provide demonstration that indeed many of these changes in the pattern of protein synthesis are induced by continued cell-cell interactions in these tight aggregates. The notion that there is a relationship between morphology and gene expression ’ Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305. * To whom reprint requests should be sent.
in Dictyostelium is not new. Newell et al. (1971, 1972) demonstrated that physical perturbation of normal development resulted in altered patterns of accumulation of several enzymatic activities. The essence of their results is that certain enzymes accumulate only when cells form tight aggregates and that continued accumulation of these enzymes depends on continued morphological integrity. Furthermore, the kinetics of accumulation of certain enzymes is dependent upon whether the aggregates form migrating slugs or proceed directly to culmination. Development in Dictyostelium is initiated by removing nutritional sources, specifically any of several amino acids (Marin, 1976). Approximately 3-4 hr after the outset of starvation, the cells become responsive to pulses of CAMP and capable of relaying the pulses (Robertson and Cohen, 1972). By 8 hr, the cells are cohesive, possess surface antigens not found in growing cells (Beug et al., 19731, and contain high levels of a carbohydrate-binding protein (Rosen et al., 1973). These changes 207
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0012-1606
208
DEVELOPMENTAL BIOLOGY
take place whether the cells are plated onto a solid surface or are kept shaking in suspension. Thus, starvation leads to the acquisition of aggregation competence (Gerisch, 1968). Once the cells have becompetent, further come aggregation changes in CAMP receptor and phosphodiesterase levels (Henderson, 1975) require that the cells be allowed to form tight aggregates. In this paper we have tested the hypothesis that synthesis of the “aggregationstage” proteins by Dictyostelium requires continuous cell contact. By studying the effects of disaggregation and reaggregation on protein synthesis, we conclude that continued cell contact is required for synthesis of a number of proteins and, presumably, for induction of transcription of the genes encoding these mRNAs. Continued cell contact is also required for cessation of synthesis of several proteins whose synthesis is characteristic of preaggregation cells. MATERIALS
AND
METHODS
All procedures for cell culture, differentiation, labeling with [35S]methionine, and analysis of radioactive proteins by electrophoresis on two-dimensional polyacrylamide gels (C)‘Farrell, 1975) are detailed in the previous paper (Alton and Lodish, 1977). In all cases cells were labeled for 15 min. Also in this paper is a discussion of the quantitation and limitations of this gel technique. Suspension cells are prepared by harvesting vegetative amoebas and resuspending them at 5 x lo6 cells/ml in MES-PDF, the normal buffer used for plating cells. These starved cultures were aerated by shaking at 22°C and at 150 rpm. Even after 20 hr, aggregates consisting of only a few cells could be seen. Disaggregated cells were prepared by washing cells from the filter pads into plating buffer. The cells were diluted to 5 x lo6 cells/ml and separated into single cells by rejected vigorous pipetting. These manipulations required less than 2 min.
VOLUME 60, 1977 RESULTS
Changes in the pattern of protein synthesis during development of DictyosteZium are discussed in detail in the previous paper (Alton and Lodish, 1977). For clarity, relevant features are reviewed here. Cells at the appropriate stage were labeled with [35S]methionine for 15 min. At the end of the label period, samples were prepared for 2-D gel electrophoresis. Labeled polypeptides were assayed by a modification of the two-dimensional gel system devised by O’Farrell (1975). The first dimension consists of isoelectric focusing in the presence of 9 M urea. The second dimension is electrophoresis in polyacrylamide gels containing SDS and an exponential gradient of polyacrylamide (Alton and Lodish, 1977). Between 4 and 10 hr, numerous changes in the pattern of protein synthesis can be detected by 2-D gel electrophoresis (Figs. 1 and 2). Nearly all of these changes occur after the cells have aggregated into discrete tight mounds (8 hr). Many new polypeptide species are synthesized by lo-hr cells which are not synthesized by cells before 7 hr (spots 43, 44, 46-50, 52-55, 57, 60, 62-69). Several other polypeptides show increased relative rates of synthesis (spots 19, 33, 45, 51, 56, 59, 61, 84). Simultaneously, several proteins show reduced rates of synthesis (spots 2-5, 13, 16, 23, 25, 31, 58) or cease being synthesized altogether (spot 70). Many other proteins show no detectable changes in the relative rate of synthesis. In order to test whether these changes in the patterns of protein synthesis and translatable mRNAs were affected by the formation of tight cell contacts which occur at 10 hr, we studied the pattern of proteins made by aggregation-competent cells. These are cells which were removed from growth medium and resuspended at 5 x lo6 cells/ml in the normal buffer used for cell plating (MES-PDF) but which were shaken at 110 rpm at 22°C. At this low cell density, shaking completely prevents the
ALTON AND LODISH
Developmentally
Regulated
Protein
Synthesis
209
FIG. 1. Two-dimensional gel of proteins synthesized by plated cells 4 hr after initiation of differentiation. Six million Dictyostelium cells were labeled with 50 $i of [35Slmethionine from 4 to 4.25 hr after initiation of differentiation. The cells and extracellular fluid were harvested, combined, and prepared for gel electrophoresis by boiling in a solution containing sodium dodecyl sulfate. The first dimension on these gels is isoelectric focusing from pH 3 (left) to pH 10 (right); the second dimension is electrophoresis in a gradient of polyacrylamide gel containing SDS. Larger proteins migrate closer to the top of the gel. The molecular weight of actin (43,000) serves as a calibration for the sizes of other proteins. The fluorogram of the gel was exposed to the equivalent of log cpm added for 24 hr. All of the details concerning the gel procedure are given in the previous paper (Alton and Lodish, 1977).
formation of cell-cell contacts. If cells are starved in suspension from between 5 and 20 hr and then plated for development, they will form aggregates in 3 hr, rather than the 8 hr normally required for differentiating cells. Thus, starved cells kept in suspension are said to be “aggregation competent” (Gerisch, 1968). A comparison of Figs. 1 and 3 shows that the pattern of protein synthesized by cells starved for 5 hr in suspension is very similar to that of cells plated for normal differentiation for 6 (or 3 or 8) hr. Synthesis continues of polypeptides characteristic of 4-hr differentiating cells (Nos. 3, 13, 21, 23, 58, and 70 at high levels; and 19, 33, 45, 51, 59, and 81 at low levels). Figure 4 shows the pattern of protein synthesis of cells starved in suspension for 20 hr. The pattern is very similar to that of
cells starved in suspension for 5 hr, although there are quantitative differences in the relative rates of synthesis of certain polypeptides. Important is the fact that these cells continue synthesis of several polypeptides (Nos. 13, 21, 30, 31, 45, 51, 58, and 70) whose synthesis is characteristic of preaggregation cells. Also of significance is the result that these aggregation-competent cells do not synthesize any of the polypeptides whose synthesis is characteristic of lo-hr postaggregation cells (for instance, polypeptides 43, 44, 46, 47, 48, 49, 50, 52-55, 57, 60, 62-69). It is concluded that cells, starved in suspension and competent to aggregate, do not exhibit most of the changes in the pattern of protein synthesis characteristic of normal late-aggregation-stage cells. They continue to synthesize a pattern of proteins characteristic
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FIG. 2. Two-dimensional gel of proteins cells were labeled 10 hr after plating.
BIOLOGY
synthesized
VOLUME
60, 1977
by cells plated for 10 hr. Same as Fig. 1, except that
FIG. 3. Two-dimensional gel of proteins synthesized by cells in suspension for 5 hr. Same as Fig. 1, except that cells were labeled after starvation for 5 hr in suspension.
of interphase or early-aggregation-stage cells. Thus suspension cells, mechanically prevented from aggregating, appear to
proceed more or less normally through the first 6 to 8 hr of differentiation, but then remain “locked” into a pattern of protein
ALTON
AND
LODISH
Developmentally
Regulated
Protein
FIG. 4. Two-dimensional gel of proteins synthesized by cells in suspension except that cells were labeled after starvation for 20 hr in suspension.
synthesis characteristic of that stage. They exhibit none of the changes which normally occur during or after late aggregation. When cells which have been starved in suspension for 5-20 hr are plated on filter pads, they aggregate within 3 hr. Concomitantly, they cease synthesis of polypeptide 70 and reduce synthesis of 13, 16, 25, 31, and 58, whose production is characteristic of normal preaggregation cells. They also begin synthesis of many (but not all) of the proteins whose synthesis is characteristic of normal lo-hr differentiating cells: peptides 43, 44, 47, 50, 54, 55, 57, 60, 63-69 (Fig. 5, Table 1). In the case of those proteins whose mRNAs could be identified by translation in a cell-free system, we showed that synthesis of these aggregation-stage polypeptides is accompanied by appearance of the homologous translatable mRNA. We conclude that the formation of tight cell-cell contacts is necessary for the induction of synthesis of these proteins and, presumably, for initiation of transcription of the homologous DNA genes.
Synthesis
211
for 20 hr. Same as Fig. 1,
Disaggregation The experiments in Fig. 6 and Table 1 show that continued cell-cell contact is essential for continued synthesis of these characteristic aggregation stage proteins. When cells are plated normally for 5 hr, then removed from the filter pads and placed in suspension at low density for 5 hr, they fail to synthesize many of the proteins which are made by normal lo-hr cells (Fig. 6). They continue synthesizing polypeptides such as 70 whose synthesis is characteristic of preaggregation cells. Even when cells which have been plated for 10 hr and which have formed tight cellcell aggregates are removed from the filter and shaken hard in suspension to disaggregate clumps of cells, they cease synthesis of many of the characteristic lo-hr aggregation-stage proteins and begin or increase synthesis of proteins characteristic of early aggregates (spots 13, 58,701 (Table 1). Polypeptides such as 43, 44, 54, 57, 66, 67, 68, and 69 are made by normal lo-hr cells in tight aggregates but not when these cells have been placed into suspen-
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VOLUME 60, 1977
FIG. 5. Two-dimensional gel of proteins synthesized by cells 5 hr after plating preceded by 5 hr in suspension. Cells harvested from growth medium were kept in suspension for 5 hr before plating. After 5 hr on plates, the cells were labeled for 15 min as described in Fig. 1. At this time the cells had formed aggregates morphologically similar to normal lo-hr differentiating cells. TABLE
1
PROTEIN SYNTHESIS IN PLATED CELLS, SUSPENSION CELLS, DISAGGREGATED CELLS AND REAGGREGATED CELL@ Protein
13 16 33 43 44 45 48 49 50 51 54 57 58 60 65 66 67 68 69 70
Plated
Suspension/ plated
Suspension
Disaggregatedl reaggregated
5
10
5
5
10
15
20
515
1015
515
IO/5
++ ++ ” 0 0 2 0 0 0 + 0 0 + 0 0 0 0 0 0 +
” + + + + + + + + + + + k + + + + + + 0
? + + + + + + + + + + + 2 + + + + + + 0
+ + k 0 0 t 0 0 0 0 0 0 + 0 0 0 0 0 0 +
+ + f 0 0 k 0 0 0 0 0 0 + 0 0 0 0 0 0 +
+ + _t 0 0 i0 0 0 0 0 0 + 0 0 0 0 0 0 +
+ + 2 0 0 + 0 0 0 0 0 0 + 0 0 0 0 0 0 +
+ + + + + + 0 + + + + + 2 + + + + + ++ 0
t 0 + + 0 + 0 0 0 + 0 + k 0 0 + + + ++ 0
L 5 + + + + -t + + 5 + + 5 + + + + + -t 0
+ k + + 0 + 0 + 0 0 + ++ t 0 + + 0 + k 0
Disaggregatedl suspension 515
+ ++ + 0 0 k 0 0 0 f 0 0 + 0 0 0 0 0 0 +
1015
+ + k 0 0 f 0 0 0 -t 0 + + 0 f ” 0 0 t +
a The numbers at the head of each column indicate the time in hours since removal of the cells from growth medium. Numbers such as 5/5 indicate that cells were either on plates or in suspension for 5 hr, then shifted to another condition for 5 hr before labeling of the cells. The 0, +, + , and + + indicate the relative rate of synthesis.
ALTON
AND LODISH
Developmentally
Regulated
Protein
Synthesis
213
FIG. 6. Two-dimensional gel of proteins synthesized by cells plated for 5 hr, disaggregated, and kept in suspension for 5 hr. Cells were allowed to differentiate normally on plates for 5 hr, then were disaggregated and starved in suspension culture for an additional 5 hr. At this point they were labeled for 15 min with [%lmethionine. Gel analysis is as in Fig. 1.
sion culture. Low levels of polypeptides 57, 66, and 69 are occasionally made by cells which have been plated for 10 hr and then kept in suspension for 5. Presumably this occurs because some mRNA encoding these proteins persists after the removal to suspension culture, but that additional mRNA encoding these proteins is not made after removal from the plates. Synthesis of protein 70 is characteristic of preaggregation but not of lo-hr plated cells. It is of interest that synthesis of this “preaggregation” protein resumes when lo-hr cells are placed in suspension (Table 1). Presumably this is due to resynthesis of mRNA encoding this protein since translatable mRNA for protein 70 is not present (or is present in low levels) in normal cells at 10 hr of differentiation (Alton and Lodish, 1977). Manipulations associated with disaggregation or reaggregation are not, in themselves, sufficient to affect the pattern of protein synthesis. If cells at 10 or 15 hr of normal differentiation are removed from
the filter, disaggregated, and then replated, they will form tight aggregates (characteristic of normal lo-hr differentiating cells) within 3 hr. The pattern of proteins made by these disaggregated and replated cells is that characteristic of normal cells (Table 1). There are some stage-specific proteins which do not fall into categories such as “aggregation induced” or “aggregation repressed.” Some proteins which are synthesized by cells at 10 hr of normal (plated) development are not synthesized by cells which are removed from filters at 5 hr after plating, dissociated, and then replated for 5 hr before labeling (spots 44,48, 51, and 601, in spite of the fact that these cells form morphologically normal aggregates. Some proteins are synthesized by aggregated cells which were plated immediately upon removal from growth medium and allowed to develop for 10 or 15 hr, but are not synthesized by aggregated cells which were kept in suspension for 5 or 10 hr and then plated for 5 hr before
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DEVELOPMENTAL BIOLOGY
labeling (spots 48 and 49). Furthermore, there are proteins which are synthesized by aggregating cells incubated for 5 hr in suspension followed by 5 hr on filters, but which are not synthesized by cells kept 10 hr in suspension followed by 5 hr on filters (peptides 44, 50, 52, 60, and 65). DISCUSSION
The principal conclusion of this work is that continued cell-cell contact is essential for synthesis of a number of developmentally regulated proteins which are characteristic of postaggregation cells. We that translatable showed previously mRNAs encoding these proteins can be detected only in postaggregation cells (Alton and Lodish, 1977). Thus continued cell contact is required for the formation of translatable mRNAs for these characteristic proteins and, presumably, for transcription of the appropriate genes. While there is no evidence for “masked” cytoplasmic mRNA species (Alton and Lodish, 1977), and there is little processing involved in formation of Dictyostelium mRNA (Lodish et al., 1973; Dottin et al., 1976; Weiner and Lodish, in preparation), we cannot eliminate the possibility that appearance of these translatable mRNAs is regulated at a post-transcriptional level. Cell Contact During
Late Aggregation
The changes in the pattern of protein synthesis which occur during the first 8 hr of normal differentiation (interphase and early aggregation) also occur in cells which have been starved in suspension instead of being plated. A few differences in the pattern of protein synthesis can be detected between plated and suspension cells, but these differences most likely reflect physiological rather than developmental differences. Cells starved in suspension show very few changes after the first 5 hr. The pattern of protein synthesis in cells kept in suspension for 20 hr is virtually indistinguishable from the pattern of protein syn-
VOLUME 80, 1977
thesis of cells in suspension for 5 hr (Figs. 2-4). These suspension cells are competent to aggregate and will do so within 2 hr after plating. They contain levels of CAMP phosphodiesterase, CAMP surface receptors, and several other surface molecules characteristic of normal aggregating cells (Beug et al., 1973; Henderson, 1975; Huesgen and Gerisch, 1975). But acquisition of the state of “aggregation competence” is insufficient to induce synthesis of mRNAs and proteins which are characteristic of aggregating cells. Cells which are plated exhibit many changes in the pattern of protein synthesis during late aggregation. During this time the cells form tight mounds of approximately lo5 cells each. Cells which have been kept in suspension and then plated show many of these same changes when the morphology of these aggregates is identical to that of a normal late aggregate. Furthermore, the changes can be prevented or even reversed by removing cells from the filter papers, dispersing the aggregates into single cells, and keeping them in suspension for 5 hr. These data lead to the hypothesis that cell contact plays an essential role in the normal developmental program. Cell contact acts by inducing transcription of new genes and repressing transcription of other genes. Furthermore, continued cell contact is essential for continued expression of the genes activated by cell contact, and for continued repression of the genes repressed by cell contact. Further insight into this problem has come from a study of mutants which are unable to aggregate (Lodish et al., 1976; Margolskee and Lodish, in preparation). The pattern of protein synthesis by two independent mutants which show no signs of aggregation was studied by two-dimensional gel electrophoresis. All such mutants show the same pattern of protein synthesis as do wild-type cells for the first 6 hr after plating. However, these mutants continue making most of the “early” pro-
ALTON
AND LODISH
Developmentally Regulated Protein Synthesis
teins, including actin, for at least 24 hr and exhibit no induction of polypeptides synthesized in wild-type cells during aggregation. Although we do not know the primary defect in any of these mutants, this work is consistent with our conclusion that cell-cell contact is required for induction of most aggregation-stage proteins. Previous work suggested that cell contact might affect expression of certain developmentally regulated genes, but the experiments were less direct than those detailed here. Mutants which are unable to aggregate generally do not accumulate the levels of activities of certain enzymes which are characteristic of aggregating cells (Loomis et al., 1976). The necessity of continuous cell contact for maintenance of the late aggregationpostaggregation state was demonstrated by Okamoto and Takeuchi (1976). Postaggregation-stage cells were disaggregated and resuspended as single cells, and the activity of two enzymes was monitored. The activity of cell-bound CAMP-bound phosphodiesterase, an enzyme characteristic of preaggregating cells, began to increase immediately, and the activity of UDP-galactose polysaccharide transferase, an activity characteristic of late differentiating cells, began to decrease immediately. Thus these disaggregated cells came to resemble cells which had not aggregated. Newell et al. (1971,1972) showed that continued cell aggregation was essential for appearance of enzymes specific for later morphological stages. The effects of cell contact on induction of new gene functions described here differ from the cases of embryonic induction of new gene functions. Embryonic induction requires generally the interaction of two different tissues, and one tissue is induced to differentiate by contact with the other. the changes in the patIn Dictyostelium tern of protein synthesis (and therefore in the pattern of gene expression) do not require the interaction of two different tissues, but rather the presence of a number
215
of cells in contact with each other. The “induction phenomenon” in slime molds is a self-induction rather than a heterologous induction. However, the mechanism of induction may well be similar in slime molds and in organogenesis in higher organisms. Smart and Tuchman (1976) demonstrated that autoclaved membranes isolated from late-aggregation- or early-postaggregation-stage cells inhibit aggregation of cells plated in the presence of these membrane preparations. Reaggregation of cells that had formed tight aggregates and then disaggregated was not inhibited by these membranes. Tuchman et al. (1976) investigated the effects of these membrane preparations on the accumulation of several developmentally regulated enzymes. The accumulation of one early enzyme, threonine deaminase, was essentially identical in membrane-treated and untreated cells, but the normal decrease in activity characteristic of later development did not occur in the treated cells. Accumulation of another early enzyme, N-acetylglucosaminidase, was completely inhibited in membrane-treated cells. Accumulation of UDPglucose-pyrophosphorylase and tyrosine transaminase (both late-aggregation enzymes) and P-glucosidase 2 (a culmination-specific enzyme) was also blocked. Thus membrane preparations from lateaggregation cells do have effects on the developmental program, and probably act primarily by preventing formation of normal cell-cell contacts. The elucidation of the factor (or factors) responsible for the cell contact effects awaits further investigation. It is not clear from our work whether direct cell contact is required or whether the concentration of a diffusible factor produced by aggregating cells must be above a critical level in order to induce the changes in the pattern of protein synthesis. This research was supported by Grant PCM7404869 A02 from the National Science Foundation. We thank Naomi Cohen and Martin Brock for ex-
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DEVEU)PMENTAL BIOLOGY
pert technical assistance and Marianne Robotham for going blind and crazy over deciphering the original draft. REFERENCES ALTON, T., and LODISH, H. F. (1977). Developmental changes in messenger RNAs and protein synthesis in Dicytostelium discoideum. Develop. Biol. 180-206. BEUG, H., KATZ, F. E., and GERISCH, G. (1973). Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum. J. Cell Biol. 56, 647-658. DOTTIN, R. P., WEINER, A. M., and LODISH, H. F. (1976). 5’ Terminal nucleotide sequences of the messenger RNAs of Dictyostelium discoideum. Cell 8, 233-244. GERISCH, G. (1968). Cell aggregation and differentiation in Dictyostelium. In “Current Topics in Developmental Biology” (A. A. Moscona and A. Monroy, eds.), Vol. 3, pp. 159-197. Academic Press, New York. HENDERSON, E. J. (1975). The cyclic adenosine 3’:5’monophosphate receptor of Dictyostelium discoi&urn. Binding characteristics of aggregation competent cells and variation of binding levels during the life cycle. J. Biol. Chem. 250, 4730-4736. HEUSCEN A., and GERISCH, G. (1975). Solubilized contact sites A from cell membranes of Dictyostelium discoideum. FEBS Lett. 56, 46-49. LODISH, H. F., FIRTEL, R. A., and JACOBSON, A. (1973). Transcription and structure of the genome of the cellular slime mold Dictyostelium discoideum. Cold Spring Harbor Symp. 38, 899-914. LODISH, H. F., ALTON, T., DOTTIN, R. P., WEINER, A. M., and MARGOL~KEE, J. P. (19761. Synthesis and translation of messenger RNA during differentiation of the cellular slime mold Dictyostelium discoideum. In “The Molecular Biology of Hormone Action” (J. Papaconstantinou, ed.), pp. 75-106. Academic Press, New York. LOOMIS, W. F., WHITE, S., and DIMOND, R. L. (1976).
VOLUME 60, 1977
A sequence of dependent stages in development of Dictyostelium discoideum. Develop. Biol. 53, 171177. MARIN, F. T. (1976). Regulation of development in Dictyostelium discoideum. I. Initiation of the growth to development transition by amino acid starvation. Develop. Biol. 48, 110-117. NEWELL, P. C., LONGLANDS, M., and SUSSMAN, M. (1971). Control of enzyme synthesis by cellular interaction during development of the cellular slime mold Dictyostelium discoideum. J. Mol. Biol. 58, 541-554. NEWELL, P. C., FRANKE, J., and SUSSMAN, M. (1972). Regulation of four functionally related enzymes during shifts in the developmental program ofDictyostelium discoideum. J. Mol. Biol. 63, 373382. O’FARRELL, P. H. (1975). High resolution two-dimensional gel analysis of proteins. J. Biol. Chem. 250, 4007-4021. OKAMOTO, K., and TAKEUCHI, I. (1976). Changes in activities of two developmentally regulated enzymes induced by lisaggregation of the pseudoplasmodia of Dictyostelium discoideum. Biochem. Biophys. Res. Commun. 72, 739-746. ROBERTSON, A., and COHEN, M. H. (1972). Control of developing fields. Annu. Rev. Biophys. Bioengineer. 1, 409-464. ROSEN, S. D., KAFKA, J. A., SIMPSON, D. L., and BARONDES, S. H. (1973). Developmentally regulated, carbohydrate-binding protein in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 70, 2554-2557. SMART, J. E., and TUCHMAN, J. (1976). Inhibition of the development of Dictyostelium discoideum by isolated plasma membranes. Develop. Biol51, 6376. TUCHMAN, J., SMART, J. E., and LODISH, H. F. (1976). Effects of differentiated membranes on the developmental program of the cellular slime mold. Develop. Biol. 51, 77-85.
