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Isolation and characterization of mutations affecting UDPG pyrophosphorylase activity in Dictyostelium discoideum
DEVELOPMENTAL
BIOLOGY
5%
169-181
(1976)
Isolation and Characterization Pyrophosphorylase Activity RANDALL L. DIMOND,' Department
of Biology,
of Mutations Affecting UDPG in Dictyostelium discoideum
PAUL A. FARNSWORTH, AND WILLIAM
University
of California,
Accepted
San Diego,
December
La Jolla,
F. LOOMIS
California
92093
15,1975
A procedure for screening large numbers of clones for an enzyme activity was used to isolate mutations which affect UDPG pyrophosphorylase activity (EC 2.7.7.9) in the cellular slime mold Dictyostelium discoideum. Five strains were recovered which have little or no UDPG pyrophosphorylase activity. Ten other strains were found which have significant activity in uiuo which is rapidly inactivated upon cell lysis. These strains have permitted us to evaluate the role of UDPG pyrophosphorylase during growth and development. The enzyme affects the growth rate of the cells but is not essential for growth. However, during development the lack of enzyme activity leads to cell death and lysis. Strains which lack UDPG pyrophosphorylase accomplish early developmental events but are unable to culminate. However, certain biochemical and cytological differentiations associated with late stages were observed. INTRODUCTION
Metabolism of polysaccharides in the development of the cellular slime mold Distyostelium discoideum is most noticeable during final fruiting body construction. Stored glycogen is broken down to glucosel-phosphate which is converted to UDP glucose by the reaction catalyzed by UDPG pyrophosphorylase (EC 2.7.7.9). UDPG, in turn, serves as a precursor for cellulose, mucopolysaccharide, and trehalose, all of which accumulate in the fruiting bodies and together account for about 13% of the dry weight (Sussman and Sussman, 1969). The specific activity of UDPG pyrophosphorylase increases dramatically during the pseudoplasmodial stage and reaches a peak at approx 19 hr. (Ashworth and Sussman, 1967). The increase in specific activity is accompanied by an increase in the differential rate of synthesis of the enzyme (Franke and Sussman, 1973; Gustafson, Kong, and Wright, 1973). A variety of mutational and environmental conditions can cause an alteration in the timing of the morphogenetic sequence or block it at various points. Many of these condiI Present address: Department chusetts Institute of Technology, sachusetts 02139.
of Biology, Cambridge,
Copyright
0 1976
Inc.
All
of reproduction
MassaMas-
tions, which affect morphogenesis prior to culmination, affect the accumulation of UDPG pyrophosphorylase (Loomis, 1975) To clarify the role of UDPG and related intermediates in development, we isolated a set of mutations which affect the activity of UDPG pyrophosphorylase. These mutant strains have confirmed that UDPG pyrophosphorylase is essential for normal cell physiology and development and have permitted us to define those processes in which it is involved. METHODS
Organism.
rights
Press,
in any form reserved.
discoideum
strain A3 has been described previously (Loomis, 1971). Strain M2 is a derivative of A3 which lacks cu-mannosidase (Free and Loomis, 19751, while strain DBL211 is a derivative of A3 which lacks N-acetylglucosaminidase (Dimond et al., 1973). The strains were grown either in broth medium (TM) (Free and Loomis, 19751, or in association with Klebsiella aerogenes and allowed to develop on membrane filters as previously described (Sussman, 1966). UDPG pyrophosphorylase assay. Cell extracts were prepared by lysing the cells in 0.1% NP-40 in an ice bath. The assay used is similar to that described by Franke
169 by Academic
Dictyostelium
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DEVELOPMENTAL BIOLOGY
and Sussman (1971). The reaction mixture contained: 1 mM UDPG; 2 mM sodium pyrophosphate; 1.6 mM NADP; lo-” M Glucose-l, 6-diphosphate; 1 miV EDTA; 4 mM MgCl,; 0.05 units phosphoglucomutase; 0.14 units glucoseS-phosphate dehydrogenase; 85 mM Tricine (pH 7.6); and the cell extract in a total volume of 1 ml. One unit of activity is defined as the amount of enzyme necessary to hydrolyse 1 nmole of UDPG per minute at 22°C. Specific activity is expressed as units of enzyme activity per milligram of total cell protein. Protein was measured by the method Lowry et al. (1951) using bovine serum albumin as a standard. Mutagenesis and cloning. Exponentially growing cells were treated with the mutagen N-methyl-N’-nitro-N-nitrosoguanidine as previously described (Dimond et al., 1973). The cells were cloned into multitest wells with a multiple pipette (Brenner et al., 1975) at a cell titer that would provide an average of one cell per well when the survivial frequency following mutagenesis was 0.1%. Screening. The inoculated trays were incubated at 22°C for 3-4 weeks to allow all viable cells to form visable clones. The trays were replicated and shifted to 27°C for 24 hr to inactivate any thermolabile enzyme. A multiple pipette was used to add 0.1 ml of the screening assay mixture to each well. It contained: 0.9 mM UDPG; 2 mM sodium pyrophosphate; 1.4 mM NADP; 2 x lo-” M glucose-l, 6-diphosphate; 2 n&f EDTA; 8 mM MgCl,; 0.28 units/ml glucose-6-phosphate dehydrogenase; 0.1 units/ml phosphoglucomutase; 80 mM Tricine (pH 7.6); 2 pg/ml phenazine methosulfate; 1 mg/ml nitro blue tetrazolium; 1% NP-40. The mauve precipitate produced in the wells containing UDPG pyrophosphorylase activity was screenable in 20 min. The trays were screened before 60 min so that background reactions did not obscure the results. Those colonies which produced the precipitate at a reduced rate were scored as putative mutant
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strains and viable cells were recovered from the replicate trays. Cells of the putative mutant strains were grown at 27°C and the UDPG pyrophosphorylase activity determined in the standard assay. Electron microscopy. Samples were fixed in the presence of ruthenium red and prepared for electron microscopy as previously described (Farnsworth and Loomis, 1975). Other enzyme assays. The assays for glycogen phosphorylase (Firtel and Bonner, 1972), alkaline phosphatase (Loomis, 19691, and P-glucosidase (Coston and Loomis, 1969), were performed as previously described. Chemicals. Phenazine methosulfonate and nitro blue tetrazolium were purchased from Calbiochem, La Jolla, Calif. RESULTS
There is sufficient UDPG pyrophosphorylase activity in axenically grown cells of Dictyostelium discoideum to permit a direct screening of cell lysates for those strains which lack activity. Mutagenized cells which had grown in multitest plates were replicated. Following replication, the original trays were shifted to 27°C for 24 hr to inactivate any thermolabile enzyme. A multiple pipette was then used to add a drop of the assay mixture in 1% NP-40 (see Methods). The cells were rapidly lysed and the activity screened within 1 hr. Those colonies which failed to produce the mauve, reduced nitro blue tetrazolium were recovered from the replica plate and subsequently screened for UDPG pyrophosphorylase activity (see Methods). A total of 15 mutant strains were recovered (Table 1) from the 18,400 colonies screened. All of the mutant strains which were recovered had low or unmeasurable activity when the standard assay was used. However, the specific activity in several strains varied from one determination to the next and was unstable in the extracts. In an attempt to estimate the in viuo activ-
DIMOND,
FARNSWORTH
AND
Mutations
LOOMIS
ity more closely, we developed a cuvettelysis assay. Whole cells were suspended in the standard reaction mixture in a cuvette, the non-ionic detergent NP-40 was added to lyse the cells, and the activity was recorded immediately following lysis. Control assays lacking one of the substrates, sodium pyrophosphate, were recorded simultaneously to correct for the absorbance change associated with the cell lysis. Ten of the strains (U2, UM5-12, UN4) were found to have significant activity when assayed by the cuvette lysis method (Table 1). The estimated activity varied between 23 and 90% of the wild-type activity. Two strains, UN2 and UN3, in which there was no detectable activity in the standard assay had low but measurable activity in the cuvette lysis assay. As shown in Fig. 1, the activity detectable in the cuvette-lysis assay was very labile following lysis. The cells were harvested and an aliquot immediately assayed using the cuvette lysis assay. The rest of the cells were lysed with NP-40 and TABLE UDPG
1
PYROPHOSPHORYLASE STRAINS
StraiV
Standard assaf (units/ w) -__-~---~--~--~__~__~__~__~__ Wild-type Ul UMl UN1 UN2 UN3 u2 UM5 UM6 UM7 UM8 UM9 UMlO UMll UM12 UN4
99.9 co.3 <0.3 10.3 CO.3 10.3 1.3 0.4 9.0 5.6 <0.3 co.3 2.0 co.3 10.3 1.5
IN THE
MUTANT
Cuvette lysis assay (units/ mg) 99.9 <1
’ Strains Ul and U2 were isolated from strain A3. The UM series was isolated from Strain M2. The UN series was isolated from strain DBL 211. b Extracts of vegetative cells were assayed.
Affecting
UDPG
Pyrophosphorylase
Activity
17
1
25
1: Ttme
2c Cller
Lysis
30
lmd
FIG. 1. Lysis-lability. The cells were harvested and an aliquot immediately assayed in the cuvette lysis assay. The rest of the cells were lysed with NP40 in an ice bath. At various times following lysis aliquots were assayed in the standard assay. The same results were obtained if the extracts were kept atroom temperature. (O-0-0) UM6; (O-0-0) UN4; (A - a - A) U2 (UM8 is identical); (X - x - X) UM5 (UM7, 9, 10, 11, 12 are identical). The solid symbols are for strain A3.
kept in an ice bath. At various times aliquots were assayed with the standard assay. In all 10 strains (U2, UM5-12, UN41 the activity decreased rapidly following lysis. The lysis-labile strains can be divided into four classes (UM6; UN4; U2, UM8; UM5, 7, 9, 10, 11, 12) on the basis of their kinetics of inactivation following lysis (Fig. 1). Mixtures of A3 wild-type cells and lysis-labile cells were assayed in the same manner. In all cases the kinetics of inactivation were identical to those expected if the A3 enzyme was totally stable and the lysis-labile enzyme inactivated with the kinetics seen in Fig. 1. Thus, the inactivation of UDPG pyrophosphorylase in the lysis-labile strains is not caused by inhibitors present in the cell extract. It is probable that the instability of UDPG pyrophosphorylase in lysis-labile strains is a property of the enzyme itself. All of the lysis-labile strains grow and develop normally. The remaining five strains (Ul, UMl, UNl-3) have very little or undetectable UDPG pyrophosphorylase activity in either assay system (Table 1). Cells from each of these strains were allowed to develop on filters and extracts were made at
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various times after the initiation of development. None of the strains contained more UDPG pyrophosphorylase activity than listed in Table 1 at any time during development. Four of the strains (Ul, UMl, UNl, 2) have identical phenotypes which are described below. Strain UN3 forms some aggregates which appear identical to the other four strains and some which culminate producing relative wellformed fruiting bodies. We assume strain UN3 has slightly more UDPG pyrophosphorylase in uivo than the others (Ul, UMl, UNl, 2) and thus develops better. Growth on bacteria. The strains which lack appreciable UDPG pyrophosphorylase grow more slowly in association with bacteria than the parental strains. The parental strains have an average generation time of 4 hr, while the mutant strains have an apparent generation time of 9 hr. This lower growth rate is at least partially caused by the inability of some of the cells to divide. When cells ofD. discoideum are cloned on bacterial growth plates a plaque develops in the bacterial lawn which enlarges radially. When cells were collected from the edge of the growth ring, the cloning efficiency (proportion of visible cells that will form plaques) of the parental strains was greater than 95%, while the cloning efficiency of strains lacking UDPG pyrophosphorylase activity was reduced to 60%. If in each cell division only 60% of the progeny continued dividing this would account for the reduced growth rate. Although the cells are unable to divide they remain metabolically active for a period of time, since they form a normal number of aggregates during development which are of approximately normal size. These aggregates continued to synthesize a variety of enzymes at the normal rate for at least 20 hr, as will be discussed below.
FIG. 2. Vegetative cells membrane whorl structure Strain Ul. Osmoregulatory
(a).
growing unique vacuole
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Examination of sections of vegetative cells by electron microscopy shows that the mutant cells have an altered osmoregulatory apparatus. Well developed, 2-Frn diameter, membrane-bound electron transparent vacuoles and an associated multivesicular system, such as seen in Fig. 2, are found in 2% of the sectioned cell profiles of the wild-type and 75% of the profiles of strain Ul. The ubiquitous observation of small vesicles fusing with the large regulatory vacuole in cells of strain Ul suggests that the rate of fusion, and thus presumably the rate of water elimination, is increased. Other aspects of cellular ultrastructure appear normal. Development of strains grown on bacteria. The early aspects of development in the strains lacking UDPG pyrophosphorylase appear normal. The cells aggregate well at low or high density, form “standing fingers” at the appropriate time, and go though a “Mexican hat” stage in assuming the proper shape to begin culmination. Under low-salt conditions in the dark, they form slugs; however, the slugs are nonmigratory and soon lose their integrity. Cellulose is a structural component of the surface sheath ofD. discoideum which imparts strength to the slug (Hohl and Jehli, 1973; Freeze and Loomis, unpublished). A component containing the cellulose fibrils can be isolated as an insoluble sheet by treatment of slugs with 9 M urea in 2% sodium dodecyl sulfate. This component makes up about 0.05% of the dry weight of wild-type slugs but is completely absent in slugs of the mutant strains lacking UDPG pyrophosphorylase. The amorphous continuous component of the surface sheath described by Farnsworth and Loomis (1975) can be seen in electron micrographs of slugs of strain Ul although it is approx 30% thinner toward the rear of
on bacteria. A: Strain A3. Mitochondria to cells feeding on bacteria (w); peripheral (V); fusing vesicle (v); structure suggesting
Cm); ingested bacterium (b) microfibrilar array (0. B: the beginning of autolysis
__.
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the slugs. Electron dense circular profiles 125 to 150 A are seen on the outward side of the homogeneous layer of the surface sheath of slugs as they are in the wildtype; however, the frequency of their occurrence was reduced by at least 95% in the slugs of strain Ul, suggesting that they are probably composed of cellulose, and represent cross sections of the fibrils
FIG.
3. Terminal
morphology.
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seen by Hohl and Jehli (1973). Under conditions where migration does not occur, the structures formed by strains lacking UDPG pyrophosphorylase activity appear normal during the first 20 hr of development. At the beginning of culmination morphogenesis becomes abnormal, as shown in Fig. 3. The slugs assume a variety of shapes ranging from collapsing
A: Strain
A3; B: Strain
Ul.
DIMOND,
FARNSWORTH
AND LOOMIS
Mutations
piles of cells to standing fingers with bulbous apical regions. By about 30 hr the apical regions begin to accumulate the yellow pigment which is also found in the sorus of the wild-type fruiting body. When viewed with the electron microscope, it is obvious that both cellular and multicellular organization is deranged in slugs and terminal “standing fingers.” The “standing finger” displayed in Fig. 4 has large regions of acellular debris which accounts for 30% or more of the volume of the organism; there is also frequent cannibalism and evidence of widespread autolysis. Most of the cells are swollen and contain many lysosomes and large degratory vacuoles of membrane fragments, partially degraded mitochondria, and other cell debris. There is no evidence of the deposition of cellulose on any cell membranes or any cellulose stalk tube formation. These observations are consistent with an inability to form cellulose due to the lack of UDPG biosynthesis in these strains. Because it was apparent that, late in development, cells which lacked UDPG pyrophosphorylase activity lysed, we determined when during development the cells lost the ability to divide. Cells of strain UMl and wild-type strain A3 were deposited on membrane filters and allowed to develop. At various times cells were washed off the filters with broth medium (TM) and titered by their ability to form plaques on bacterial growth plates. The data presented in Fig. 5 is normalized to the number of plaque-forming cells per filter when the cells were first deposited. It should be remembered that in these vegetative cells the cloning efficiency of the wild-type cells was lOO%, while that of the UMl cells was only 15%. The apparent loss of cells able to form plaques in strain A3 (Fig. 5) was due to difficulties in recovery from the filters and in an inability to completely dissociate the cells. If the cells are triturated in EDTA, the apparent recovery is much higher due to better separation of cells, but we wanted to avoid harsh treatment of the UMl cells which were known
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Pyrophosphorylase
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to be fragile. In contrast to the wild-type A3 cells, cells of strain UMl rapidly lost the ability to divide and form plaques. By 40 hr of development, the number of cells able to divide had been reduced by more than five orders of magnitude. Evidence of cytodifferentiation. Although the cells of strain Ul and UMl eventually die and lyse, there is evidence that some of the cells proceeded through some of the early steps in spore differentiation. Figure 6 shows examples of crenelate mitochondrial profiles, cytoplasmic condensation, the studding of internal membranes by ribosomes, and the disappearance of prespore vesicles. These changes are diagnostic of, and unique to, spore differentiation (George et al., 1972). However, the cells do not become elipsoidal and there is no evidence of any cell-wall formation. The cell walls of mature spores are known to contain cellulose (Raper and Fennell, 1952), and would not be expected to form in these strains. There appears to be no organized spatial pattern to such cytodifferentiation, in contrast to the marked separation of cell types in the parent strains (Fig. 4). It is difficult to judge if any of the cells proceed toward stalk differentiation; most of the cells become swollen, vacuolize, and accumulate the protein crystals commonly seen in stalk cell formation. However, such properties are not unique to stalk cell differentiation and may represent stages in the loss of cellular organization leading to cell death. True stalk cells are dead, but during their terminal vacuolization they form thick rigid angular walls of cellulose fibers. Strains Ul and UMl do not show any evidence of cellulose thickening of the cell walls of these swollen cells. Biochemical differentiation. Because some of the cells in strains lacking UDPG pyrophosphorylase activity were clearly proceeding towards spore differentiation, we investigated the appearance of enzymes which are specific to the culmination stage of development. Figure 7 demonstrates that the accumulation of glyco-
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DIMOND,
FARNSWORTH
AND
Mutations
LOOMIS
d
01
I
\
L
1 Ld
1 IO
031
20
30
40
d n Time (hrsi
5. Plaque-forming cells per filter during development. Cells were deposited on filters to initiate development. At various times cells were washed off the filters and the titer determined by a plaque assay on bacterial growth plates. The results are normalized to the number of plaque-forming cells per filter initially. CO---•lA3; (A - A)UMl. FIG.
A
Affecting UDPG Pyrophosphorylase
Activity
177
blocked morphologically at preculmination, it is clear that there is no block in the program that leads to biochemical differentiation (Loomis et al., 1976). Synergy. It is common in D. discoideum to find that cells of morphologically aberrant strains will synergize and form normal fruiting bodies if they are allowed to develop with wild-type cells or with cells of a different morphological mutant strain. This appears to be the case at the gross morphological level but not at the level of cell differentiation with strains lacking UDPG pyrophosphorylase activity. When cells lacking this activity are mixed with wild-type cells such that the wild-type cells represent 5% of the population, a large number of normally proportioned fruiting bodies are formed. Even when the wild-type cells represent only 1% of the population, a few fruiting bodies are formed. The stalk portion of these fruiting bodies appears normal although the sorus contains swollen, vacuolated cells, cell debris, and only a few spores. All of the cells from such sori which are able to grow on bacteria are, however, wild-type. Thus, although the cells lacking UDPG pyrophosphorylase are assisted by the wild-type cells in forming fruiting bodies, they are still unable to form spores or avert death.
gen phosphorylase and alkaline phosphatase late in the development is normal in strains lacking UDPG pyrophosporylase activity. It is interesting that the peak activity of alkaline phosphatase occurs at a time when the number of plaque-forming cells has been reduced to 0.1% of the original (compare Fig. 5). Although the cells have lost the ability to divide they are still DISCUSSION functional in protein synthesis. The accumulation of P-glucosidase-2, the latest The selection of mutations which alter a known stage-specific enzyme, was reduced known gene is a powerful tool in defining in the mutant strains although the activ- those functions for which the gene product ity was electrophoretically identified as p- is required. In the cellular slime mold, glucosidase-2. The peak specific activity of Dictyostelium discoideum, it has proven /3-glucosidase-2 normally occurs approx 2 feasible to directly screen for mutant hr after the peak of alkaline phosphatase. strains which have reduced activity for The reduced synthesis of P-glucosidase-2 specific enzymes (Dimond et al., 1973; Free in cells lacking UDPG pyrophosphorylase and Loomis, 1975). Utilizing this apactivity probably results from cell death proach, we have isolated 15 strains with and lysis. Although the mutant strains are altered UDPG pyrophosphorylase activity. FIG. angular
4. Terminal cytodifferentiation. A: Strain A3 during late culmination. Vacuolated cellulose wall (w); cellulosic stalk tube CT); undifferentiated amoeba (A); condensed process of sporulation (D); mature spore (~1. B: Strain Ul from a similar time in development. of tissue organization. The cell mass is mostly cellular debris (B), swollen autolysing undifferentiated cells with fusing vacuolar system W).
stalk cell (c); amoeba in the Note the lack cells (S), and
178
DIMOND.
FARNSWORTH
AND LOOMIS
Mutations
Affecting
UDPG
Pyrophosphorylase
Activity
179
that if we were able to sufficiently mimic the in vivo environment we could stablize the activity. Even the wild-type enzyme itself is not stable following lysis, and care \ / CL , has to be taken to preserve its activity (Newell and Sussman 1969; Pannbacker 1967). Perhaps a high proportion of alterations in the protein structure produce an enzyme with pronounced instability. The instability of the enzyme from lysislabile strains has complicated the interP-I pretation of the mutant strains in which there is no detectable activity. It is impossible to eliminate the possibility that these 18 24 30 36 strains actually contain some activity in lLa.6 avivo, but that it is so unstable in vitro as to Time ihrsi be unmeasurable. To limit these possibiliFIG. 7. Late stage specific enzymes. Cells were allowed to develop on membrane filters. At various ties we have looked for the enzymatic times the cells were collected in 3 ml of distilled product UDPG and saccharides for which water and assayed. A: Glycogen phosphorylase; B: it is the precursor and have found that the alkaline phosphatase; C: P-glucosidase (O-•IA3; UDPG pyrophosphorylase activity is (A-A-A, Ul. drastically reduced in vivo in these strains In three of the strains we have been unaalthough it may not be eliminated. ble to detect any enzyme activity, while in There are suggestions in the literature two additional strains the activity is re- that UDPG pyrophosphorylase present in duced approx 98%. vegetative cells may differ from the enThe remaining 10 strains are novel in zyme in developing cells (Gustafson et al ., that, while they contain significant UDPG 1973), and that the enzyme is polymorphic pyrophosphorylase activity in vivo, this (Newell and Sussman, 1969). Since all of activity is rapidly inactivated upon cell the activity is affected at all times during lysis. We have referred to these strains as development in the mutant strains it is lysis-labile strains since we have been unclear that although there may be some able to find conditions of lysis, buffer conheterogeneity in the form of the enzyme it ditions, or additives which will stabilize is likely there is only a single UDPG pyrthe activity. The loss of activity does not ophosphorylase gene. At least some of the appear to be due to inhibitors or degradamutations appear to have modified the tive enzymes in the extracts since mixstructural gene for UDPG pyrophosphoryltures of wild-type and mutant extracts ase in that we isolated four distinct classes showed no effect of the mutant extract on of strains in which the enzyme is lysisthe activity of the wild-type enzyme. Thus, labile as well as the three null mutants. It we believe that the lysis-lability is a propis highly unlikely that this enzyme is suberty of the enzyme itself and that these ject to five distinct post-translational modstrains may carry mutations in the strucifications affecting the activity. Genetic tural gene for this enzyme. We assume mapping and complementation studies FIG. 6. Cytodifferentiation (n). B: Crystalline deposit deposit which is apparently ribosomes.
in strain Ul. similar to those membrane-bound
A: Autolytic vacuoles found in degenerating (x1. C: Crenulate
(VI; mitochondrion (ml; nuclear region stalk cells of the wild-type (y); crystal mitochondria which are studded with
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BIOLOGY
may further define the number of alleles. The loss of enzyme activity has drastic effects on the organism both at the levels of cellular physiology and multicellular morphogenesis. The cells appear to have difficulty with osmoregulation at all stages of growth and differentiation and a reduced viability. The cells develop large autodegradative vacuoles and cannibalism becomes common. By 22-24 hr after the removal of nutrients, the cells are unable to compensate for the loss of UDPG pyrophosphorylase activity and begin to swell and lyse. It is interesting that although the mutant cells appear abnormal at all times they function effectively in morphogenesis for at least 20 hr. In the developmental pathway leading to fruiting body formation, morphogenesis appears normal until the start of culmination. At that point morphogenesis is arrested presumably because of the inability to form cellulose. It is also possible that morphogenesis is arrested as a result of cellular degradation. Under conditions that favor slug formation, slugs are formed in the mutant strains; however, they are nonfunctional and soon lose integrity. Again, the inability to form the cellulose sheath around the organism may contribute to the lack of ability of the slugs to migrate. Although the loss of UDPG pyrophosphorylase activity prevents the formation of saccharides that depend upon UDPG as a precursor and arrests those stages of morphogenesis that require cellulose, all prior morphogenetic events are normal. The strains that lack UDPG pyrophosphorylase activity continue the normal course of expression of stage-specific enzymes including those that peak at the end of culmination. The organisms also accumulate the yellow pigment that appears long after culmination is complete. Some of the cells are able to complete most of the cytoplasmic differentiations associated with spore formation. It seems clear that although multicellularity is required for
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the biochemical differentiation of late development there is no requirement for any specific topological arrangement of the cells. We thank Wayne Borth for technical assistance. This work was supported by grants from the National Institutes of Health (GM 19543-03) and the National Science Foundation (GB 28955). REFERENCES ASHWORTH, J. M., and SUSSMAN, M. (1967). The appearance and disappearance of uridine diphosphate glucose pyrophosphorylase activity during differentiation of the cellular slime mold Dictyostelium discoideum. J. Biol. Chem. 242, 1696-1700. BRENNER, M., TISDALE, D., and LOOMIS, W. F., JR. (1975). Techniques for rapid biochemical screening of large numbers of cell clones. Exp. Cell Res. 90, 249. COSTON, M. B., and LOOMIS, W. F. (1969). Isozymes of P-glucosidase in Dictyostelium discoideum. J. Bact. 100, 1208-1217. DIMOND, R. L., BRENNER, M., and LOOMIS, W. F. (1973). Mutations affecting N-acetylglucosaminidase in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 70, 3356. FARNSWORTH, P. A., and LOOMIS, W. F. (1975). A gradient in the thickness of t.he surface sheath in pseudoplasmedia of Dictyostelium discoideum. Dev. Biol. 46, 349-357. FIRTEL, R. A., and BONNER, J. (1972). Developmental control of a-1-4 glucan phosphorylase in the cellular slime mold Dictyostelium discoideum. J. Deu. Biol. 29, 85-103. FRANKE, J., and SUSSMAN, M. (1971). Synthesis of uridine diphosphate glucose pyrophosphorylase during the development of Dictyostelium discoideum. J. Biol. Chem. 246, 6381-6388. FRANKE, J., and SUSSMAN, M. (1973). Accumulation of uridine diphosphoglucose pyrophosphatase in Dictyostelium discoidenm via preferential synthesis. d. Mol. Biol. 81, 173-185. FREE, S., and LOOMIS, W. F. (1975). Isolations of mutations in Dictyostelium discoideum affecting ru-mannosidase. Biochimie 56, 1525-1528. GEORGE, R., HOHL, H., and RAPER, K. (1972). Ultrastructural development of stalk-producing cells in Dictyostelium discoideum, a cellular slime mould. J. Gen. Microbial. 70, 477-489. GUSTAFSON, G. L., KONG, W. Y., and WRIGHT, B. E. (1973). Analysis of uridine diphosphate-glucose pyrophosphorylase synthesis during differentiation in Dictyostelium discoideum. J. Biol. Chem. 248, 5188-5196. HOHL, H. R., and JEHLI, J. (1973). The presence of cellulose microfibrils in the proteinaceous slime track of Dictyostelium discoideum. Arch. Mikrobiol. 92, 179-187.
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FARNSWORTH
AND LOOMIS
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LOOMIS, W. F. (1969). Developmental regulation of alkaline phosphatase in Dictyostelium discoideum. J. Bact. 100, 417-422. LOOMIS, W. F. (1971). Sensitivity of Dictyostelium discoideum to nucleic acid analogues. Exp. Cell Res. 64, 484-486. LOOMIS, W. F. (1975). “Dictyostelium discoideum A Developmental System,” p. 126. Academic Press, New York. LOOMIS, W. F., DIMOND, R. L., FREE, S. L., and WHITE, S. (in press). “Eukaryotic Microbes as Model Developmental Systems” (D. O’Day and P. Horgen, eds.). LOWRY, 0. H., ROSEBROUGH, N., FARR, A., and RANDALL, R. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. NEWELL, P. C., and SUSSMAN, M. (1969). Uridine diphosphate glucose pyrophosphorylase in Dictyostelium discoideum. Stability and developmental fate. J. Biol. Chem. 244, 2990-2995.
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PANNBACKER, R. G. (1967). Uridine diphosphoglucase biosynthesis during differentiation in the cellular slime mold. In vitro measurements. Biochemistry 6, 1287-1293. RAPER, K. B., and FENNELL, D. I. (1952). Stalk formation in Dictyostelium. Bull. Torrey B&an. Club 79, 25-51. SUSSMAN, M., and SUSSMAN, R. R. (19691. Patterns of RNA synthesis and of enzyme accumulation and disappearance during cellular slime mold cytodifferentiation. Symp. Sot. Gen. Microbial. 19, 403-435. SUSSMAN, M. (1966). Biochemical and genetic methods in the study of cellular slime mold development. In “Methods in Cell Physiology” (D. Prescott, ed.1 Vol. 2, p. 397. Academic Press, New York. WRIGHT, B. E., and DAHLBERG, D. (1967). Cell wall synthesis in Dictyostelium discoideum. II. Synthesis of soluble glycogen by a cytoplasmic enzyme. Biochemistry 6, 2074-2079.
BIOLOGY
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Isolation and Characterization Pyrophosphorylase Activity RANDALL L. DIMOND,' Department
of Biology,
of Mutations Affecting UDPG in Dictyostelium discoideum
PAUL A. FARNSWORTH, AND WILLIAM
University
of California,
Accepted
San Diego,
December
La Jolla,
F. LOOMIS
California
92093
15,1975
A procedure for screening large numbers of clones for an enzyme activity was used to isolate mutations which affect UDPG pyrophosphorylase activity (EC 2.7.7.9) in the cellular slime mold Dictyostelium discoideum. Five strains were recovered which have little or no UDPG pyrophosphorylase activity. Ten other strains were found which have significant activity in uiuo which is rapidly inactivated upon cell lysis. These strains have permitted us to evaluate the role of UDPG pyrophosphorylase during growth and development. The enzyme affects the growth rate of the cells but is not essential for growth. However, during development the lack of enzyme activity leads to cell death and lysis. Strains which lack UDPG pyrophosphorylase accomplish early developmental events but are unable to culminate. However, certain biochemical and cytological differentiations associated with late stages were observed. INTRODUCTION
Metabolism of polysaccharides in the development of the cellular slime mold Distyostelium discoideum is most noticeable during final fruiting body construction. Stored glycogen is broken down to glucosel-phosphate which is converted to UDP glucose by the reaction catalyzed by UDPG pyrophosphorylase (EC 2.7.7.9). UDPG, in turn, serves as a precursor for cellulose, mucopolysaccharide, and trehalose, all of which accumulate in the fruiting bodies and together account for about 13% of the dry weight (Sussman and Sussman, 1969). The specific activity of UDPG pyrophosphorylase increases dramatically during the pseudoplasmodial stage and reaches a peak at approx 19 hr. (Ashworth and Sussman, 1967). The increase in specific activity is accompanied by an increase in the differential rate of synthesis of the enzyme (Franke and Sussman, 1973; Gustafson, Kong, and Wright, 1973). A variety of mutational and environmental conditions can cause an alteration in the timing of the morphogenetic sequence or block it at various points. Many of these condiI Present address: Department chusetts Institute of Technology, sachusetts 02139.
of Biology, Cambridge,
Copyright
0 1976
Inc.
All
of reproduction
MassaMas-
tions, which affect morphogenesis prior to culmination, affect the accumulation of UDPG pyrophosphorylase (Loomis, 1975) To clarify the role of UDPG and related intermediates in development, we isolated a set of mutations which affect the activity of UDPG pyrophosphorylase. These mutant strains have confirmed that UDPG pyrophosphorylase is essential for normal cell physiology and development and have permitted us to define those processes in which it is involved. METHODS
Organism.
rights
Press,
in any form reserved.
discoideum
strain A3 has been described previously (Loomis, 1971). Strain M2 is a derivative of A3 which lacks cu-mannosidase (Free and Loomis, 19751, while strain DBL211 is a derivative of A3 which lacks N-acetylglucosaminidase (Dimond et al., 1973). The strains were grown either in broth medium (TM) (Free and Loomis, 19751, or in association with Klebsiella aerogenes and allowed to develop on membrane filters as previously described (Sussman, 1966). UDPG pyrophosphorylase assay. Cell extracts were prepared by lysing the cells in 0.1% NP-40 in an ice bath. The assay used is similar to that described by Franke
169 by Academic
Dictyostelium
170
DEVELOPMENTAL BIOLOGY
and Sussman (1971). The reaction mixture contained: 1 mM UDPG; 2 mM sodium pyrophosphate; 1.6 mM NADP; lo-” M Glucose-l, 6-diphosphate; 1 miV EDTA; 4 mM MgCl,; 0.05 units phosphoglucomutase; 0.14 units glucoseS-phosphate dehydrogenase; 85 mM Tricine (pH 7.6); and the cell extract in a total volume of 1 ml. One unit of activity is defined as the amount of enzyme necessary to hydrolyse 1 nmole of UDPG per minute at 22°C. Specific activity is expressed as units of enzyme activity per milligram of total cell protein. Protein was measured by the method Lowry et al. (1951) using bovine serum albumin as a standard. Mutagenesis and cloning. Exponentially growing cells were treated with the mutagen N-methyl-N’-nitro-N-nitrosoguanidine as previously described (Dimond et al., 1973). The cells were cloned into multitest wells with a multiple pipette (Brenner et al., 1975) at a cell titer that would provide an average of one cell per well when the survivial frequency following mutagenesis was 0.1%. Screening. The inoculated trays were incubated at 22°C for 3-4 weeks to allow all viable cells to form visable clones. The trays were replicated and shifted to 27°C for 24 hr to inactivate any thermolabile enzyme. A multiple pipette was used to add 0.1 ml of the screening assay mixture to each well. It contained: 0.9 mM UDPG; 2 mM sodium pyrophosphate; 1.4 mM NADP; 2 x lo-” M glucose-l, 6-diphosphate; 2 n&f EDTA; 8 mM MgCl,; 0.28 units/ml glucose-6-phosphate dehydrogenase; 0.1 units/ml phosphoglucomutase; 80 mM Tricine (pH 7.6); 2 pg/ml phenazine methosulfate; 1 mg/ml nitro blue tetrazolium; 1% NP-40. The mauve precipitate produced in the wells containing UDPG pyrophosphorylase activity was screenable in 20 min. The trays were screened before 60 min so that background reactions did not obscure the results. Those colonies which produced the precipitate at a reduced rate were scored as putative mutant
VOLUME 50. 1976
strains and viable cells were recovered from the replicate trays. Cells of the putative mutant strains were grown at 27°C and the UDPG pyrophosphorylase activity determined in the standard assay. Electron microscopy. Samples were fixed in the presence of ruthenium red and prepared for electron microscopy as previously described (Farnsworth and Loomis, 1975). Other enzyme assays. The assays for glycogen phosphorylase (Firtel and Bonner, 1972), alkaline phosphatase (Loomis, 19691, and P-glucosidase (Coston and Loomis, 1969), were performed as previously described. Chemicals. Phenazine methosulfonate and nitro blue tetrazolium were purchased from Calbiochem, La Jolla, Calif. RESULTS
There is sufficient UDPG pyrophosphorylase activity in axenically grown cells of Dictyostelium discoideum to permit a direct screening of cell lysates for those strains which lack activity. Mutagenized cells which had grown in multitest plates were replicated. Following replication, the original trays were shifted to 27°C for 24 hr to inactivate any thermolabile enzyme. A multiple pipette was then used to add a drop of the assay mixture in 1% NP-40 (see Methods). The cells were rapidly lysed and the activity screened within 1 hr. Those colonies which failed to produce the mauve, reduced nitro blue tetrazolium were recovered from the replica plate and subsequently screened for UDPG pyrophosphorylase activity (see Methods). A total of 15 mutant strains were recovered (Table 1) from the 18,400 colonies screened. All of the mutant strains which were recovered had low or unmeasurable activity when the standard assay was used. However, the specific activity in several strains varied from one determination to the next and was unstable in the extracts. In an attempt to estimate the in viuo activ-
DIMOND,
FARNSWORTH
AND
Mutations
LOOMIS
ity more closely, we developed a cuvettelysis assay. Whole cells were suspended in the standard reaction mixture in a cuvette, the non-ionic detergent NP-40 was added to lyse the cells, and the activity was recorded immediately following lysis. Control assays lacking one of the substrates, sodium pyrophosphate, were recorded simultaneously to correct for the absorbance change associated with the cell lysis. Ten of the strains (U2, UM5-12, UN4) were found to have significant activity when assayed by the cuvette lysis method (Table 1). The estimated activity varied between 23 and 90% of the wild-type activity. Two strains, UN2 and UN3, in which there was no detectable activity in the standard assay had low but measurable activity in the cuvette lysis assay. As shown in Fig. 1, the activity detectable in the cuvette-lysis assay was very labile following lysis. The cells were harvested and an aliquot immediately assayed using the cuvette lysis assay. The rest of the cells were lysed with NP-40 and TABLE UDPG
1
PYROPHOSPHORYLASE STRAINS
StraiV
Standard assaf (units/ w) -__-~---~--~--~__~__~__~__~__ Wild-type Ul UMl UN1 UN2 UN3 u2 UM5 UM6 UM7 UM8 UM9 UMlO UMll UM12 UN4
99.9 co.3 <0.3 10.3 CO.3 10.3 1.3 0.4 9.0 5.6 <0.3 co.3 2.0 co.3 10.3 1.5
IN THE
MUTANT
Cuvette lysis assay (units/ mg) 99.9 <1
’ Strains Ul and U2 were isolated from strain A3. The UM series was isolated from Strain M2. The UN series was isolated from strain DBL 211. b Extracts of vegetative cells were assayed.
Affecting
UDPG
Pyrophosphorylase
Activity
17
1
25
1: Ttme
2c Cller
Lysis
30
lmd
FIG. 1. Lysis-lability. The cells were harvested and an aliquot immediately assayed in the cuvette lysis assay. The rest of the cells were lysed with NP40 in an ice bath. At various times following lysis aliquots were assayed in the standard assay. The same results were obtained if the extracts were kept atroom temperature. (O-0-0) UM6; (O-0-0) UN4; (A - a - A) U2 (UM8 is identical); (X - x - X) UM5 (UM7, 9, 10, 11, 12 are identical). The solid symbols are for strain A3.
kept in an ice bath. At various times aliquots were assayed with the standard assay. In all 10 strains (U2, UM5-12, UN41 the activity decreased rapidly following lysis. The lysis-labile strains can be divided into four classes (UM6; UN4; U2, UM8; UM5, 7, 9, 10, 11, 12) on the basis of their kinetics of inactivation following lysis (Fig. 1). Mixtures of A3 wild-type cells and lysis-labile cells were assayed in the same manner. In all cases the kinetics of inactivation were identical to those expected if the A3 enzyme was totally stable and the lysis-labile enzyme inactivated with the kinetics seen in Fig. 1. Thus, the inactivation of UDPG pyrophosphorylase in the lysis-labile strains is not caused by inhibitors present in the cell extract. It is probable that the instability of UDPG pyrophosphorylase in lysis-labile strains is a property of the enzyme itself. All of the lysis-labile strains grow and develop normally. The remaining five strains (Ul, UMl, UNl-3) have very little or undetectable UDPG pyrophosphorylase activity in either assay system (Table 1). Cells from each of these strains were allowed to develop on filters and extracts were made at
172
DEVELOPMENTAL
BIOLOGY
various times after the initiation of development. None of the strains contained more UDPG pyrophosphorylase activity than listed in Table 1 at any time during development. Four of the strains (Ul, UMl, UNl, 2) have identical phenotypes which are described below. Strain UN3 forms some aggregates which appear identical to the other four strains and some which culminate producing relative wellformed fruiting bodies. We assume strain UN3 has slightly more UDPG pyrophosphorylase in uivo than the others (Ul, UMl, UNl, 2) and thus develops better. Growth on bacteria. The strains which lack appreciable UDPG pyrophosphorylase grow more slowly in association with bacteria than the parental strains. The parental strains have an average generation time of 4 hr, while the mutant strains have an apparent generation time of 9 hr. This lower growth rate is at least partially caused by the inability of some of the cells to divide. When cells ofD. discoideum are cloned on bacterial growth plates a plaque develops in the bacterial lawn which enlarges radially. When cells were collected from the edge of the growth ring, the cloning efficiency (proportion of visible cells that will form plaques) of the parental strains was greater than 95%, while the cloning efficiency of strains lacking UDPG pyrophosphorylase activity was reduced to 60%. If in each cell division only 60% of the progeny continued dividing this would account for the reduced growth rate. Although the cells are unable to divide they remain metabolically active for a period of time, since they form a normal number of aggregates during development which are of approximately normal size. These aggregates continued to synthesize a variety of enzymes at the normal rate for at least 20 hr, as will be discussed below.
FIG. 2. Vegetative cells membrane whorl structure Strain Ul. Osmoregulatory
(a).
growing unique vacuole
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50. 1976
Examination of sections of vegetative cells by electron microscopy shows that the mutant cells have an altered osmoregulatory apparatus. Well developed, 2-Frn diameter, membrane-bound electron transparent vacuoles and an associated multivesicular system, such as seen in Fig. 2, are found in 2% of the sectioned cell profiles of the wild-type and 75% of the profiles of strain Ul. The ubiquitous observation of small vesicles fusing with the large regulatory vacuole in cells of strain Ul suggests that the rate of fusion, and thus presumably the rate of water elimination, is increased. Other aspects of cellular ultrastructure appear normal. Development of strains grown on bacteria. The early aspects of development in the strains lacking UDPG pyrophosphorylase appear normal. The cells aggregate well at low or high density, form “standing fingers” at the appropriate time, and go though a “Mexican hat” stage in assuming the proper shape to begin culmination. Under low-salt conditions in the dark, they form slugs; however, the slugs are nonmigratory and soon lose their integrity. Cellulose is a structural component of the surface sheath ofD. discoideum which imparts strength to the slug (Hohl and Jehli, 1973; Freeze and Loomis, unpublished). A component containing the cellulose fibrils can be isolated as an insoluble sheet by treatment of slugs with 9 M urea in 2% sodium dodecyl sulfate. This component makes up about 0.05% of the dry weight of wild-type slugs but is completely absent in slugs of the mutant strains lacking UDPG pyrophosphorylase. The amorphous continuous component of the surface sheath described by Farnsworth and Loomis (1975) can be seen in electron micrographs of slugs of strain Ul although it is approx 30% thinner toward the rear of
on bacteria. A: Strain A3. Mitochondria to cells feeding on bacteria (w); peripheral (V); fusing vesicle (v); structure suggesting
Cm); ingested bacterium (b) microfibrilar array (0. B: the beginning of autolysis
__.
173
174
DEVELOPMENTAL
BIOLOGY
the slugs. Electron dense circular profiles 125 to 150 A are seen on the outward side of the homogeneous layer of the surface sheath of slugs as they are in the wildtype; however, the frequency of their occurrence was reduced by at least 95% in the slugs of strain Ul, suggesting that they are probably composed of cellulose, and represent cross sections of the fibrils
FIG.
3. Terminal
morphology.
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50,
1976
seen by Hohl and Jehli (1973). Under conditions where migration does not occur, the structures formed by strains lacking UDPG pyrophosphorylase activity appear normal during the first 20 hr of development. At the beginning of culmination morphogenesis becomes abnormal, as shown in Fig. 3. The slugs assume a variety of shapes ranging from collapsing
A: Strain
A3; B: Strain
Ul.
DIMOND,
FARNSWORTH
AND LOOMIS
Mutations
piles of cells to standing fingers with bulbous apical regions. By about 30 hr the apical regions begin to accumulate the yellow pigment which is also found in the sorus of the wild-type fruiting body. When viewed with the electron microscope, it is obvious that both cellular and multicellular organization is deranged in slugs and terminal “standing fingers.” The “standing finger” displayed in Fig. 4 has large regions of acellular debris which accounts for 30% or more of the volume of the organism; there is also frequent cannibalism and evidence of widespread autolysis. Most of the cells are swollen and contain many lysosomes and large degratory vacuoles of membrane fragments, partially degraded mitochondria, and other cell debris. There is no evidence of the deposition of cellulose on any cell membranes or any cellulose stalk tube formation. These observations are consistent with an inability to form cellulose due to the lack of UDPG biosynthesis in these strains. Because it was apparent that, late in development, cells which lacked UDPG pyrophosphorylase activity lysed, we determined when during development the cells lost the ability to divide. Cells of strain UMl and wild-type strain A3 were deposited on membrane filters and allowed to develop. At various times cells were washed off the filters with broth medium (TM) and titered by their ability to form plaques on bacterial growth plates. The data presented in Fig. 5 is normalized to the number of plaque-forming cells per filter when the cells were first deposited. It should be remembered that in these vegetative cells the cloning efficiency of the wild-type cells was lOO%, while that of the UMl cells was only 15%. The apparent loss of cells able to form plaques in strain A3 (Fig. 5) was due to difficulties in recovery from the filters and in an inability to completely dissociate the cells. If the cells are triturated in EDTA, the apparent recovery is much higher due to better separation of cells, but we wanted to avoid harsh treatment of the UMl cells which were known
Affecting
UDPG
Pyrophosphorylase
Activity
175
to be fragile. In contrast to the wild-type A3 cells, cells of strain UMl rapidly lost the ability to divide and form plaques. By 40 hr of development, the number of cells able to divide had been reduced by more than five orders of magnitude. Evidence of cytodifferentiation. Although the cells of strain Ul and UMl eventually die and lyse, there is evidence that some of the cells proceeded through some of the early steps in spore differentiation. Figure 6 shows examples of crenelate mitochondrial profiles, cytoplasmic condensation, the studding of internal membranes by ribosomes, and the disappearance of prespore vesicles. These changes are diagnostic of, and unique to, spore differentiation (George et al., 1972). However, the cells do not become elipsoidal and there is no evidence of any cell-wall formation. The cell walls of mature spores are known to contain cellulose (Raper and Fennell, 1952), and would not be expected to form in these strains. There appears to be no organized spatial pattern to such cytodifferentiation, in contrast to the marked separation of cell types in the parent strains (Fig. 4). It is difficult to judge if any of the cells proceed toward stalk differentiation; most of the cells become swollen, vacuolize, and accumulate the protein crystals commonly seen in stalk cell formation. However, such properties are not unique to stalk cell differentiation and may represent stages in the loss of cellular organization leading to cell death. True stalk cells are dead, but during their terminal vacuolization they form thick rigid angular walls of cellulose fibers. Strains Ul and UMl do not show any evidence of cellulose thickening of the cell walls of these swollen cells. Biochemical differentiation. Because some of the cells in strains lacking UDPG pyrophosphorylase activity were clearly proceeding towards spore differentiation, we investigated the appearance of enzymes which are specific to the culmination stage of development. Figure 7 demonstrates that the accumulation of glyco-
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DEVELOPMENTAL
BIOL~CY
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50, 1976
DIMOND,
FARNSWORTH
AND
Mutations
LOOMIS
d
01
I
\
L
1 Ld
1 IO
031
20
30
40
d n Time (hrsi
5. Plaque-forming cells per filter during development. Cells were deposited on filters to initiate development. At various times cells were washed off the filters and the titer determined by a plaque assay on bacterial growth plates. The results are normalized to the number of plaque-forming cells per filter initially. CO---•lA3; (A - A)UMl. FIG.
A
Affecting UDPG Pyrophosphorylase
Activity
177
blocked morphologically at preculmination, it is clear that there is no block in the program that leads to biochemical differentiation (Loomis et al., 1976). Synergy. It is common in D. discoideum to find that cells of morphologically aberrant strains will synergize and form normal fruiting bodies if they are allowed to develop with wild-type cells or with cells of a different morphological mutant strain. This appears to be the case at the gross morphological level but not at the level of cell differentiation with strains lacking UDPG pyrophosphorylase activity. When cells lacking this activity are mixed with wild-type cells such that the wild-type cells represent 5% of the population, a large number of normally proportioned fruiting bodies are formed. Even when the wild-type cells represent only 1% of the population, a few fruiting bodies are formed. The stalk portion of these fruiting bodies appears normal although the sorus contains swollen, vacuolated cells, cell debris, and only a few spores. All of the cells from such sori which are able to grow on bacteria are, however, wild-type. Thus, although the cells lacking UDPG pyrophosphorylase are assisted by the wild-type cells in forming fruiting bodies, they are still unable to form spores or avert death.
gen phosphorylase and alkaline phosphatase late in the development is normal in strains lacking UDPG pyrophosporylase activity. It is interesting that the peak activity of alkaline phosphatase occurs at a time when the number of plaque-forming cells has been reduced to 0.1% of the original (compare Fig. 5). Although the cells have lost the ability to divide they are still DISCUSSION functional in protein synthesis. The accumulation of P-glucosidase-2, the latest The selection of mutations which alter a known stage-specific enzyme, was reduced known gene is a powerful tool in defining in the mutant strains although the activ- those functions for which the gene product ity was electrophoretically identified as p- is required. In the cellular slime mold, glucosidase-2. The peak specific activity of Dictyostelium discoideum, it has proven /3-glucosidase-2 normally occurs approx 2 feasible to directly screen for mutant hr after the peak of alkaline phosphatase. strains which have reduced activity for The reduced synthesis of P-glucosidase-2 specific enzymes (Dimond et al., 1973; Free in cells lacking UDPG pyrophosphorylase and Loomis, 1975). Utilizing this apactivity probably results from cell death proach, we have isolated 15 strains with and lysis. Although the mutant strains are altered UDPG pyrophosphorylase activity. FIG. angular
4. Terminal cytodifferentiation. A: Strain A3 during late culmination. Vacuolated cellulose wall (w); cellulosic stalk tube CT); undifferentiated amoeba (A); condensed process of sporulation (D); mature spore (~1. B: Strain Ul from a similar time in development. of tissue organization. The cell mass is mostly cellular debris (B), swollen autolysing undifferentiated cells with fusing vacuolar system W).
stalk cell (c); amoeba in the Note the lack cells (S), and
178
DIMOND.
FARNSWORTH
AND LOOMIS
Mutations
Affecting
UDPG
Pyrophosphorylase
Activity
179
that if we were able to sufficiently mimic the in vivo environment we could stablize the activity. Even the wild-type enzyme itself is not stable following lysis, and care \ / CL , has to be taken to preserve its activity (Newell and Sussman 1969; Pannbacker 1967). Perhaps a high proportion of alterations in the protein structure produce an enzyme with pronounced instability. The instability of the enzyme from lysislabile strains has complicated the interP-I pretation of the mutant strains in which there is no detectable activity. It is impossible to eliminate the possibility that these 18 24 30 36 strains actually contain some activity in lLa.6 avivo, but that it is so unstable in vitro as to Time ihrsi be unmeasurable. To limit these possibiliFIG. 7. Late stage specific enzymes. Cells were allowed to develop on membrane filters. At various ties we have looked for the enzymatic times the cells were collected in 3 ml of distilled product UDPG and saccharides for which water and assayed. A: Glycogen phosphorylase; B: it is the precursor and have found that the alkaline phosphatase; C: P-glucosidase (O-•IA3; UDPG pyrophosphorylase activity is (A-A-A, Ul. drastically reduced in vivo in these strains In three of the strains we have been unaalthough it may not be eliminated. ble to detect any enzyme activity, while in There are suggestions in the literature two additional strains the activity is re- that UDPG pyrophosphorylase present in duced approx 98%. vegetative cells may differ from the enThe remaining 10 strains are novel in zyme in developing cells (Gustafson et al ., that, while they contain significant UDPG 1973), and that the enzyme is polymorphic pyrophosphorylase activity in vivo, this (Newell and Sussman, 1969). Since all of activity is rapidly inactivated upon cell the activity is affected at all times during lysis. We have referred to these strains as development in the mutant strains it is lysis-labile strains since we have been unclear that although there may be some able to find conditions of lysis, buffer conheterogeneity in the form of the enzyme it ditions, or additives which will stabilize is likely there is only a single UDPG pyrthe activity. The loss of activity does not ophosphorylase gene. At least some of the appear to be due to inhibitors or degradamutations appear to have modified the tive enzymes in the extracts since mixstructural gene for UDPG pyrophosphoryltures of wild-type and mutant extracts ase in that we isolated four distinct classes showed no effect of the mutant extract on of strains in which the enzyme is lysisthe activity of the wild-type enzyme. Thus, labile as well as the three null mutants. It we believe that the lysis-lability is a propis highly unlikely that this enzyme is suberty of the enzyme itself and that these ject to five distinct post-translational modstrains may carry mutations in the strucifications affecting the activity. Genetic tural gene for this enzyme. We assume mapping and complementation studies FIG. 6. Cytodifferentiation (n). B: Crystalline deposit deposit which is apparently ribosomes.
in strain Ul. similar to those membrane-bound
A: Autolytic vacuoles found in degenerating (x1. C: Crenulate
(VI; mitochondrion (ml; nuclear region stalk cells of the wild-type (y); crystal mitochondria which are studded with
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DEVELOPMENTAL
BIOLOGY
may further define the number of alleles. The loss of enzyme activity has drastic effects on the organism both at the levels of cellular physiology and multicellular morphogenesis. The cells appear to have difficulty with osmoregulation at all stages of growth and differentiation and a reduced viability. The cells develop large autodegradative vacuoles and cannibalism becomes common. By 22-24 hr after the removal of nutrients, the cells are unable to compensate for the loss of UDPG pyrophosphorylase activity and begin to swell and lyse. It is interesting that although the mutant cells appear abnormal at all times they function effectively in morphogenesis for at least 20 hr. In the developmental pathway leading to fruiting body formation, morphogenesis appears normal until the start of culmination. At that point morphogenesis is arrested presumably because of the inability to form cellulose. It is also possible that morphogenesis is arrested as a result of cellular degradation. Under conditions that favor slug formation, slugs are formed in the mutant strains; however, they are nonfunctional and soon lose integrity. Again, the inability to form the cellulose sheath around the organism may contribute to the lack of ability of the slugs to migrate. Although the loss of UDPG pyrophosphorylase activity prevents the formation of saccharides that depend upon UDPG as a precursor and arrests those stages of morphogenesis that require cellulose, all prior morphogenetic events are normal. The strains that lack UDPG pyrophosphorylase activity continue the normal course of expression of stage-specific enzymes including those that peak at the end of culmination. The organisms also accumulate the yellow pigment that appears long after culmination is complete. Some of the cells are able to complete most of the cytoplasmic differentiations associated with spore formation. It seems clear that although multicellularity is required for
VOLUME
50, 1976
the biochemical differentiation of late development there is no requirement for any specific topological arrangement of the cells. We thank Wayne Borth for technical assistance. This work was supported by grants from the National Institutes of Health (GM 19543-03) and the National Science Foundation (GB 28955). REFERENCES ASHWORTH, J. M., and SUSSMAN, M. (1967). The appearance and disappearance of uridine diphosphate glucose pyrophosphorylase activity during differentiation of the cellular slime mold Dictyostelium discoideum. J. Biol. Chem. 242, 1696-1700. BRENNER, M., TISDALE, D., and LOOMIS, W. F., JR. (1975). Techniques for rapid biochemical screening of large numbers of cell clones. Exp. Cell Res. 90, 249. COSTON, M. B., and LOOMIS, W. F. (1969). Isozymes of P-glucosidase in Dictyostelium discoideum. J. Bact. 100, 1208-1217. DIMOND, R. L., BRENNER, M., and LOOMIS, W. F. (1973). Mutations affecting N-acetylglucosaminidase in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 70, 3356. FARNSWORTH, P. A., and LOOMIS, W. F. (1975). A gradient in the thickness of t.he surface sheath in pseudoplasmedia of Dictyostelium discoideum. Dev. Biol. 46, 349-357. FIRTEL, R. A., and BONNER, J. (1972). Developmental control of a-1-4 glucan phosphorylase in the cellular slime mold Dictyostelium discoideum. J. Deu. Biol. 29, 85-103. FRANKE, J., and SUSSMAN, M. (1971). Synthesis of uridine diphosphate glucose pyrophosphorylase during the development of Dictyostelium discoideum. J. Biol. Chem. 246, 6381-6388. FRANKE, J., and SUSSMAN, M. (1973). Accumulation of uridine diphosphoglucose pyrophosphatase in Dictyostelium discoidenm via preferential synthesis. d. Mol. Biol. 81, 173-185. FREE, S., and LOOMIS, W. F. (1975). Isolations of mutations in Dictyostelium discoideum affecting ru-mannosidase. Biochimie 56, 1525-1528. GEORGE, R., HOHL, H., and RAPER, K. (1972). Ultrastructural development of stalk-producing cells in Dictyostelium discoideum, a cellular slime mould. J. Gen. Microbial. 70, 477-489. GUSTAFSON, G. L., KONG, W. Y., and WRIGHT, B. E. (1973). Analysis of uridine diphosphate-glucose pyrophosphorylase synthesis during differentiation in Dictyostelium discoideum. J. Biol. Chem. 248, 5188-5196. HOHL, H. R., and JEHLI, J. (1973). The presence of cellulose microfibrils in the proteinaceous slime track of Dictyostelium discoideum. Arch. Mikrobiol. 92, 179-187.
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FARNSWORTH
AND LOOMIS
Mutations
LOOMIS, W. F. (1969). Developmental regulation of alkaline phosphatase in Dictyostelium discoideum. J. Bact. 100, 417-422. LOOMIS, W. F. (1971). Sensitivity of Dictyostelium discoideum to nucleic acid analogues. Exp. Cell Res. 64, 484-486. LOOMIS, W. F. (1975). “Dictyostelium discoideum A Developmental System,” p. 126. Academic Press, New York. LOOMIS, W. F., DIMOND, R. L., FREE, S. L., and WHITE, S. (in press). “Eukaryotic Microbes as Model Developmental Systems” (D. O’Day and P. Horgen, eds.). LOWRY, 0. H., ROSEBROUGH, N., FARR, A., and RANDALL, R. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. NEWELL, P. C., and SUSSMAN, M. (1969). Uridine diphosphate glucose pyrophosphorylase in Dictyostelium discoideum. Stability and developmental fate. J. Biol. Chem. 244, 2990-2995.
Affecting
UDPG
Pyrophosphorylase
Activity
181
PANNBACKER, R. G. (1967). Uridine diphosphoglucase biosynthesis during differentiation in the cellular slime mold. In vitro measurements. Biochemistry 6, 1287-1293. RAPER, K. B., and FENNELL, D. I. (1952). Stalk formation in Dictyostelium. Bull. Torrey B&an. Club 79, 25-51. SUSSMAN, M., and SUSSMAN, R. R. (19691. Patterns of RNA synthesis and of enzyme accumulation and disappearance during cellular slime mold cytodifferentiation. Symp. Sot. Gen. Microbial. 19, 403-435. SUSSMAN, M. (1966). Biochemical and genetic methods in the study of cellular slime mold development. In “Methods in Cell Physiology” (D. Prescott, ed.1 Vol. 2, p. 397. Academic Press, New York. WRIGHT, B. E., and DAHLBERG, D. (1967). Cell wall synthesis in Dictyostelium discoideum. II. Synthesis of soluble glycogen by a cytoplasmic enzyme. Biochemistry 6, 2074-2079.