Developmental regulation of the enzyme UDP-galactose polysaccharide transferase

© 1967 by Academic Press Inc.

328

Experimental Cell Research 46, 328-33~ (1967)

DEVELOPMENTAL GALACTOSE

REGULATION

OF THE

POLYSACCHARIDE

ENZYME

TRANSFERASE

UDP1

K . Y A N A G I S A W A 2, W. F . L O O M I S , J R . 3, and M. S U S S M A N

Brandeis University, Waltham, Mass., U.S.A. Received October 5, 1966

THE enzyme UDP-Gal polysaccharide transferase, absent in vegetative amoebae of the slime mold Dictgostelium discoideum, appears some time after multicellular aggregates have formed and accumulates to a peak of specific activity during construction of the fruiting body. It is then preferentially released into the extracellular space and is rapidly destroyed or inactivated [13, 14]. Both accumulation and disappearance require concomitant protein synthesis and a prior period of RNA synthesis [10, 11]. That this enzyme is developmentally regulated has been suggested by the performances of 3 mutant strains which display morphogenetic abnormalities [8, 16]. One of them, strain Agg-204, cannot aggregate and after growth the amoebae remain as separate individuals. Another, strain FR-2, can aggregate but stops development at this point, the component cells retaining their amoeboid form. Neither strain accumulates detectible transferase activity [14]. A third mutant, strain FR-17, is temporally deranged, reaching its terminal stage of development in about half the time required by the wild type to construct fruits. The terminal stage is a flattened, irregular, papillated aggregate within which the amoebae differentiate into normal spores and stalk cells. In accord with the overall acceleration of morphogenesis, a variety of end and by products detectible by biochemical and immunochemical assays have been found to appear sooner and to accumulate more rapidly than in the wild type. UDP-Gal transferase is also formed and disappears precociously in this strain. In the present study, the performance of five other morphogenetically deficient mutant strains further supports the conclusion that the transferase is developmentally regulated. The steps in the pattern of control appear to be correlated with specific morphogenetic events in such a w a y that (a) a 1 2 (T1 a

This work was supported by a grant from the National Science Foundation (GB 1310). Postdoctoral Trainee (Graduate Training Program) in Developmental Biology for NIPI HD-22). Present address: Department of Biology, University of California, San Diego, Calif., U.S.A.

Experimental Cell Research 46

The enzyme UDP-galactose polgsaccharide transferase

329

m u t a n t strain i n c a p a b l e of d e v e l o p i n g b e y o n d a p a r t i c u l a r m o r p h o l o g i c a l stage is also i n c a p a b l e of a p p l y i n g those controls over U D P - G a l t r a n s f e r a s e activity w h i c h the wild type brings into p l a y d u r i n g s u b s e q u e n t d e v e l o p m e n t ; ( b ) w h e n deficient m u t a n t s u n d e r g o synergistic m o r p h o g e n e s i s [9], the synergism extends to the control of t r a n s f e r a s e activity.

METHODS

Organisms and conditions of cxperiment.--D, discoideum NC-4 (haploid) and mutant derivatives were grown on SM medium in association with Aerobacler aerogenes [12]. When exponential growth had ceased, after about 40-44 hr of incubation, the cells were harvested in cold water, washed 3 times by eentrifugation for 5 rain at 1200 × g, suspended in water at 2 × 10 s eells/ml and distributed in 0.5 ml aliquots on black 2 inch millipores. The millipores rested on absorbent pads saturated with buffersalt-streptomycin solution inside 60 m m petri dishes. The incubation temperature was 22°C. Development is highly synchronous under these conditions. For enzyme assays, the cells from single millipores were harvested in 3 ml of Tris (0.01 M), thioglycolate (5 × 103 M), p H 7.5 buffer and frozen. Immediately before assay, they were thawed and broken in a Branson Sonifier (intensity level = 2 amps; time = 45 see). Transferase assay.--A detailed description is given elsewhere [14]. The reaction mixture contained a buffer-salt solution, extract, UPD-l~C-galaetose (1400 epm/ re#mole) and a standard concentration of mueopolysaccharide acceptor. After 1 hr incubation at 30°C, the mixture was heated in 0.05 M HC1 for 10 rain at 100°C to hydrolyze the remaining UDP-galaetose, precipitated with 80 per cent ethanol, deposited on a millipore, washed with 80 per cent ethanol and counted at 35 per cent efficiency in a gas flow counter. Enzyme activity was measured by the aeeeptor dependent transfer of 14C-galactose into the mucopolysaccharide fraction (cpm/hr/mg protein). In active preparations the incorporation of galactose into the alcoholinsoluble fraction in the absence of acceptor was about 5 per cent of that in its presence, probably due to endogenous mucopolysaccharide. Protein determination.--This was performed by the method of Lowry et aI. [6].

RESULTS

Induction of m u t a n t s w i t h nitrosoguanidine N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (NG) has been s h o w n to be a r e m a r k a b l y potent m u t a g e n for bacteria [1, 7]. It is e q u a l l y so for the cellular slime molds. A m o e b a e were h a r v e s t e d f r o m g r o w t h plates, w a s h e d three times with 0.05 M p o t a s s i u m p h o s p h a t e buffer p H 6 b y centrifugation in the cold, s u s p e n d e d in this buffer c o n t a i n i n g 1 m g / m l NG 1 a n d i n c u b a t e d for 1 I t is v i t a l t h a t t h e N G be s t o r e d d r y a n d i n ± h e cnid a n d t h a t fresh s o l u t i o n s be p r e p a r e d for each t r e a t m e n t . E v e n if s t o r e d in t h e m a n n e r described, color c h a n g e s ( a n d t h e loss of p o t e n c y ) occur after t w o or t h r e e m o n t h s .

Experimenlal Cell Research 46

330

K. Yanagisawa, W. F. Loomis, Jr. and M. Sussman

Fig. 1.--Photomicrographs of KY-3. Left: pseudoplasmodia (15 ×). Right: aerial filaments produced during pseudoplasmodial migration (ca 15 x).

20-30 min at 22°C, T h e cells were t h e n s p u n down, w a s h e d twice with buffer, diluted a p p r o p r i a t e l y a n d plated on SM agar with bacteria. At a survival rate of 10-20 per cent, a b o u t 1 out of 15 of the resulting clones d i s p l a y e d a stable m o r p h o g e n e t i e a n o m a l y . M o s t of these m u t a n t strains w e r e either u n a b l e to aggregate or u n a b l e to f o r m m a t u r e fruits similar to m u t a n t t y p e s p r e v i o u s l y o b s e r v e d as a result of s p o n t a n e o u s m u t a t i o n or after UV irr a d i a t i o n [16]. T w o novel classes of m u t a n t s were also observed. One class. consists of strains w h i c h develop b e y o n d aggregation to p r o d u c e n o r m M p s e u d o p l a s m o d i a b u t r e m a i n in this f o r m w i t h o u t completing d e v e l o p m e n t . T h e other class consists of strains w h i c h give rise to spherical aggregate~ within w h i c h practically all the cells t r a n s f o r m into spores without evidence: of stalk formation.

Description of mutant strains emploged in the transferase sludg Strain KY-28 f o r m s perfectly s m o o t h colonies on growth m e d i u m w i t h o u t a n y sign of aggregation. Likewise, w a s h e d cells i n c u b a t e d on millipore: filters r e m a i n as a s m o o t h l a w n of a m o e b a e . Experimental Cell Research 46

The enzgme UDP-galaclose polgsaccharide trunsferase

331

Strains KY-11 a n d KY-5 f o r m i r r e g u l a r papill:ated aggregates b o t h on growth m e d i u m a n d on millipores. Strain K Y - 1 3 f o r m s n o r m a l aggregates a n d these t r a n s f o r m into upright, finger-like p s e u d o p l a s m o d i a .

z 3000

i

i

{ 77-'

",

.

KY-I[÷WlLD TYPE

°°tj'

? ....

/

}1 ....

~UTANT DEVELOPMENT ~

HOURS ~ ~9 z ~

Fig. 2.

50

25

HOURS

a

io 20 NUMBER OF WILD Type CELLS IN MfXTUREX jO-7

Fig. 3,

Fig. 4.

Fig. 2.--Developmental kinetics of UDP-Gal transferase activity in mutant KY-3 (4) and wild type D. discoideum (0). Fig. 3.--Transferase synthesis and destruction during synergistic development by mixtures of KY-11 and wild type amoebae (cell ratio, 10:1, respeetively). Fig. 4.--Relation between peak scientific transferase activity accumulated and the number of wild type present in synergistic mixtures with 10s KY-13 amoebae. Strain KY-3 f o r m s n o r m a l aggregates a n d these t r a n s f o r m into migrating slugs. F u r t h e r d e v e l o p m e n t on b o t h growth plates andl millipores is d e p e n dent on p o p u l a t i o n density. At 10 s cells/millipore, no if u r t h e r d e v e l o p m e n t occurs. At slightly lower densities, as the slugs migrat!e they spin out v e r y thin viscous material in a m a n n e r r e m i n i s c e n t of stalk f o r m a t i o n in D. mucoroides [2]. This results in the construction of fine aerial filaments terminated b y small, nippled, p e a r - s h a p e d p s e u d o p l a s m o d i a . At still lower densities, 4× 106-1× 107 cells/millipore, just a b o v e the m i n i m u m cell density r e q u i r e d for aggregation to occur, viable spores are p r o d u e e d at the termini. T h e s e b r e e d true on replating. Fig. 1 shows photo m i c r o g r a p h s of the migrating slugs a n d the aerial filaments. I

Developmental kinetics of UDP-Gal transferase in the mutants Strains K Y - I 1, KY-13 a n d KY-28 were s e p a r a t e l y i n c u b a t e d on millipores. Extracts of the m u t a n t strains h a r v e s t e d d u r i n g the p e r i o d in w h i c h the wild type a c c u m u l a t e s p e a k t r a n s f e r a s e activity, c o n t a i n e d only a few per cent of the p e a k t r a n s f e r a s e activity f o u n d in wild type cells (see Fig. 3 a n d T a b l e I for data). Strain KY-3 was f o u n d to a c c u m u l a t e t r a n s f e r a s e activity at the usual rate a n d to a level only slightly below that of the wild type (Fig. 2). H o w e v e r this activity did not s u b s e q u e n t l y d i s a p p e a r as it does in the wild Experimental Cell Research 46

332

K. Yanagisawa, W. F. Loomis, Jr. and M. Sussman

type. Furthermore, when duplicate cell samples were centrifuged at 2,500 × g for 5 min and the cell pellets separated from the supernatants, all of the enzyme activity was found to be associated with the cells, none with the supernatants. In contrast, it has been previously shown that shortly after the peak activity is attained in the wild type, at least 80 per cent of the enzyme is TABLE I. Transferase accumulation during synergistic development. Strains

Specific activity at 22 hr (as % of peak wild t y p e activity)

KY-5 KY-11 KY-28 KY-5 + KY-28 K Y - 5 + KY-11 KY-28 + KY-11

5 5 4 32 17 42

W a s h e d a m o e b a e were mixed at cell ratios of 1:1 and incubated at 108 cells/millipore. The cells were h a r v e s t e d after 22 hr at 22 °. Specific activities of transferase are c o m p a r e d w i t h the peak aetivity attained by 10 s wild t y p e cells u n d e r comparable conditions.

released into the supernatant at a specific activity 30-fold higher than in the cells I131. Thus, while strain KY-3, at the population density employed, ean aeeumulate transferase activity it can neither extrude the enzyme nor inactivate it.

Developmental kinetics of the enzgme during synergistic development W h e n cells of each of the 3 mutant strains KY-11, KY-13 and KY-28 were mixed with small numbers o f wild type amoebae (cell ratios of 10:1 and 5 : 1) and incubated on millipores, normal, mature fruiting bodies appeared. Synergistic development of this kind has been previously explored I3, 4], but not at the bioehemieal level. All three eombinations aeeumulated considerable amounts of transferase activity, far more than could be accounted for by the number of wild type cells present. Fig. 3 shows the time course of transferase activity in strain KY-11 alone and in a 10:1 mixture with wild type cells. Fig. 4 shows the peak aetivities accumulated by mixtures of 108 eells of strain KY-13 with different numbers of wild type amoebae. Strains KY-5, KY-11 and KY-28 when paired with each other in 1:1 cell ratios and incubated (108 eells/millipore) also developed synergistically. Unfortunately the level of synergism is not easily reproducible but in general, combinations of strains KY-11 and KY-5 and combinations of strains Experimenlal Cell Research 46

The enzgme UDP-galactose polgsaccbaride tra±isferase

333

KY-5 and KY-28 yielded incomplete and aberrant fruits while combinations of strains KY-11 and KY-28 yielded normal, mature fruits. When the level of synergistic development was high, considerable transferase activity accumulated as shown in Table I. DISCUSSION As mentioned previously UDP-Gal polysaccharide transferase first appears in wild type D. discoideum when cell aggregates are transformed into pseudoplasmodia, accumulates to a peak at a late stage of fruit construction, is preferentially released by the cells, and then is rapidly destroyed. A study of 8 mutant strains, here and elsewhere [141 permit the following statements: (1). Strain FR-17 proceeds through the flow of morphogenetic events nlore rapidly than the wild type. The pattern of control over transferase activity in strain FR-17 while remaining qualitatively the same as in the wild type is correspondingly accelerated. (2) Strains FR-2, Agg-204, KY-28, KY-5 and KY-11 stop development at stages prior to that which is normally accompanied by the appearance of the transferase in the wild type. Mutant strain KY-13 stops shortly thereafter. All of these strains accumulate little or no transferase activity. (3) Strain KY-3 develops to a late pseudoplasmodial stage. This, in the wild type, marks the period of transferase accumulation but is prior to attainment of peak activity as well as enzyme extrusion and inactivation. In strain KY-3, transferase activity appears at the usual time and accumulates at the normal rate to a level somewhat less than in the wild type but is neither extruded nor inactivated. (4) Strains KY-11, KY-13 and KY-28 can develop synergistically when mixed with small numbers of wild type ceils. Under these conditions, they accumulate the normal level of transferase and then destroy it. Mutant strains KY--5, KY-11 and KY-28 can develop synergistically when paired with each other. Here too, appreciable levels of transferase activity accumulate. The correlation between changes in transferase activity or location and the attainment of specific morphogenetic states cannot be taken to imply a m a n d a t o r y coupling between them but rather as the reflection of parallel events controlled by a common source. This stems from the finding that wild type cells permitted a period of undisturbed development (coinciding with the formation of early aggregates) and then dissociated and incubated under conditions which prevent reaggregation, can nevertheless accumulate the normal level of transferase activity and destroy it at the usual times while remaining as a smooth lawn of amoebae [5]. 22 - 671815

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334

K. Yanagisawa, W. F. Loomis, Jr. and M. Sussman SUMMARY

During development of Dictgostelium discoideum wild type, the enzyme UDP-galactose polysaccharide transferase appears and accumulates to a peak of specific activity, is preferentially released by the cells, and is then destroyed. These events are correlated with the occurrence of specific morphogenetic events. A comparative study of morphogenetically deficient mutants has revealed that (a) strains incapable of proceeding beyond a particular developmental stage are likewise incapable of applying those controls over transferase activity which the wild type exerts during the subsequent development; (b) mutants which ordinarily accumulate little or no transferase activity can synthesize considerable levels of enzyme when they develop synergistically in mixtures with other mutants or with the wild type. A highly effective procedure for induction of slime mold mutants using nitrosoguanidine is described. REFERENCES

1. ADELBERG, E. A., MANDELL, M. and CHEN G., Biochem. Biophgs. Res. Comm. 18, 788 (1965). 2. BONNER, J. T., Quart. Rev. Biol. 32, 232 (1957). 3. ENNIS, H. L. and SUSSMAN, M., J. Gem Microbiol. 18, 433 (1958). 4. KAHN, A. J., Develop. Biol. 9, 1 (1964). 5. Loo~Is, W. F. JR. and SUSSMAN, M., J. Molec. Biol. In press (1966). 6. LowRY, O. H., ROSEBOROUGH, N. J., FAHR, A. L. and I:~ANDALL,1:~. J., J. Biol. Chem. 193, 265 (1951). 7. M&NDELL, J. D. and GREENBERG, J., Biochem. Biophgs. Res. Comm. 13, 575 (1960). 8. SONNEBORN, D. I~., WHITE, G. J. and Sussman, M., Develop. Biol. 7, 79 (1963). 9. SUSS~AN, M., J. Gen. Microbiol. 10, 110 (1954). 10. SUSSMAN,M., Biochem. Biophys. Res. Comm. 18, 763, 7 (1965). 11. - Proc. Nail Adac. Sci. 55, 813 (1966). 12. - in D. PRESCOTT (ed.), Methods in Cell Physiology, voh 2, pp. 397-409. Academic Press, New York, 1966. 13. SUSSMAN,M. and LSVGREN, N., Exptl Cell Res. 28, 97 (1965). 14. SUSSMAN,M. and OSBORN, M. J., Proc. Natl Acad. Sci. 52, 81 (1964). 15. SUSSr~AN, M. and SUSS~IAN, t~. YI., Bioehim. Biophgs. Acta 108, 463 (1965). 16. SUSS~AN, R. R. and SUSSMAN, M., Ann. N. Y. Acad. Sci. 56, 949 (1953).

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