Cyclic nucleotides and cyclic nucleotide phosphodiesterase during development of Polysphondylium violaceum

Printed in Sweden Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/79/100265-07$02.00/O

Experimental Cell Research 122 (1979) 265-271

CYCLIC

NUCLEOTIDES

AND

PHOSPHODIESTERASE

CYCLIC

DURING

OF POL YSPHONDYLIUM MICHAEL Department

H. HANNA,’ of Biology,

CLAUDETTE Princeton

NUCLEOTIDE

DEVELOPMENT VIOLACEUM

KLEIN2 and EDWARD C. COX

University,

Princeton,

NJ 08.540, USA

SUMMARY is shown to produce and excrete cyclic nucleotides and to produce a cell-associated cyclic nucleotide phosphodiesterase(s). The amount of adenosine 3’,5’-cyclic monophosphate (CAMP) excreted by the amebae reaches a maximum during development when aggregation centers are just forming and then falls off rapidly. Measurements of total CAMP show that the amount synthesized increases more than 15-fold throughout development with the majority of the increase coming during the culmination stages. Guanosine 3’,5’-cyclic monophospbate (cGMP) is either not excreted or is excreted at levels below our limits of detection. An increase in the total cGMP synthesized occurs at mid-aggregation when two or three sharp peaks of synthesis are observed. However, development of P. violaceum is not affected by the addition of high concentrations of either CAMP or cGMP (or their dibutyryl derivatives) to the medium despite the fact that the cells produce these nucleotides. Cell-associated cyclic nucleotide phosphodiesterase activity, which hydrolyses both CAMP and cGMP, is greatest at the onset of starvation with a second increase in activity during aggregation.

Polysphondylium

violaceum

A number of cellular slime molds have been shown to excrete CAMP [l] and to produce CAMP phosphodiesterase [l, 21. Many of these species use CAMP as their chemoattractant (acrasin). Polysphondylium violaceum excretes CAMP and CAMP phosphodiesterase, but does not use CAMP as its chemoattractant. What then is the function of CAMP and CAMP phosphodiesterase in P. violaceum? Recent experiments of Town et al. [3] and Kay et al. [4] in Dictyostelium discoideum show that CAMP and other ‘factors’ can induce the differentiation of both stalk and prespore cells under the appropriate conditions. Their experiments extend the initial observations of Bonner [5] and Chia [6] on the induction of stalk cell differentiation with CAMP. These data 18-791811

suggest that CAMP may play an additional role in slime mold development, that of a morphogen. Experiments by Francis [‘ii], Hohl et al. [S] and George [9] inP. p~~~~d~rn suggest that cAMP may be an effective morphogen in species not using cAMP as their acrasin. Hohl and collaborators an George were able to stimulate stalk cell formation in aggregates of P. pallidurn with high concentrations of CAMP (1W They also showed that the apical dominance of slugs put on agar containing high concentrations of CAMP was disturbed. 1 Present address: Biology Department, Rensselaer Polytechnic Institute, Troy, NY 12181, USA. 2 Present address: Department of Biochemistry, St Louis University School of Medicine, St Louis, MG 63119, USA. Exp Cell Res 122 (1979)

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Hanna, Klein and Cox

These slugs developed a series of leg-like protuberances containing some differentiated stalk cells. Spore cells did not seem to be induced by CAMP in P. pallidurn. Although there are differences in the effects of CAMP on development in Dictyostelium and Polysphondylium species, it seems likely that CAMP plays a primary role in both cell differentiation and pattern formation [ 10-143. cGMP has been shown to be an important component of the chemotactic system in species using CAMP as their chemoattractant [IS-171 and in species such as P. violaceum [18] and D. lacteum [15] that have chemoattractants other than CAMP. Folic acid, a chemotactic substance for vegetative D. discoideum amebae, also elicits an increase in cGMP [17]. It thus seems that transient increases in cGMP may be a general part of the chemotactic response in cellular slime molds. In this paper we report the amounts of cGMP and CAMP synthesized during P. violaceum development, the intracellular and extracellular location of these nucleotides and their possible modulation by a cyclic nucleotide phosphodiesterase(s). This study complements the results of Wurster et al. [18], who measured cGMP synthesis in P. violaceum during a limited period of development.

MATERIALS

AND METHODS

Growth conditions P. violaceum no. 1 was m-own in association with Escherichia coli B/r on 5% agar plates containing 0.2% glucose plus 1X VB salts [19]. All growth plates were incubated at 22°C in the dark. Under these conditions P. vioZacettm grows with a doubling time of 34 h.

Starvation and development conditions Amebae were harvested from plates while still in vegetative growth. The amebae were pelleted by Exp CellRes 122 (1979)

centrifueation at 700 sr for 10 min and washed twice with 1% Bonnem’ salts solution [20] to remove bacteria. The amebae were resuspended in LPS buffer [21] and allowed to develop under one of the following conditions. (A) Approx. lo8 amebae were spread uniformly on prewashed dialysis membrane (15 in. diameter. Thomas) stretched over polvcarbonate drums [22]. The drums were put in covered glass dishes containing sterile LPS up to the level of and just touching the dialysis membrane (approx. 22 ml). The chambers were incubated at 22°C in the dark. (B) The cleaned amebae were resuspended in LPS buffer at 1-2X lo7 amebae/mI. Five ml of this suspension was gently filtered on to nrewashed 0.45 wrn Millipore filters (Type HA, 47 mm). The filters were placed on abosrbent nads saturated with LPS and incubated at 22°C in the-dark.

Sample collection and assay for extracellular cyclic nucleotides Amebae were allowed to develop on drums at 22°C. At 16h intervals the drums were shifted into fresh LPS buffer and reincubated. Samples of the fluid from under the drum were frozen at -20°C until assayed. The samples were thawed and the amount of CAMP or cGMP was determined by radioimmune assay (RIA) [23] using the RIA reagents from New England Nuclear. The assay procedure was slightly modified as described previously [24]. For both the CAMP and cGMP assays, as little as l-2 pmol/drum can be detected.

Sample collection and assay for total cyclic nucleotides Amebae were allowed to develop on Millipore filters at 22°C. At 1 h intervals one filter was removed and placed, amebae side down, on 1 ml of 5% trichloroacetic acid (TCA) in a 60X15 mm mastic Petri dish. Before inverting the filter, [aH]cAMP or r3H]cGMP (approx. 0.3 pmol, 2000 cpm) was added to the filter as a marker to determine recoveries. The samples were frozen in the TCA. At the end of the experiment, the samples were thawed. the TCA solution adjusted to 0.1 N HCl and extracted several times with watersaturated diethyl either. Residual ether was removed by blowing N, over the samples. The pH of each sample was adjusted to 7.0, and recoveries were determined by counting an aliquot in a toluene-Triton X-100 scintillation fluid. The amounts of CAMP and cGMP were determined by radioimmune assay, as previously described, and corrected for recovery [24].

Sample collection and assay for cyclic nucleotide phosphodiesterase Amebae were allowed to develop on Milhpore filters at 22°C. At 1; h intervals the amebae were scraped from the filters and washed in LPS buffer. The oellet was frozen at -20°C until assayed. The assa> for cellular CAMP phosphodiesterase was that described by Brooker et al. [25] as modified by Klein [26]. The assay for cellular cGMP phosphodiesterase was the

CAMP und cGA4P modulation same except that cGMP and LDH]cGMP (New England Nuclear, 8.28 Ciimmole) were substituted for CAMP and [3H]cAMP (New England Nuclear, 36.7 Ci/ mmole). The rate of hvdrolvsis was constant for 2030 min at 30°C. Units of activity are defined as pmol CAMP (or cGMP) converted to S’AMP (or S’GMP) per mg ‘protein per min at 30°C. Protein‘was deter: mined by the method of Lowry et al. [27].

in P. violaceum

ofdevelopment

2

16 1

Effect of added cyclic nucleotides on development CAMP, cGMP, db-CAMP (W, 02’-dibutyryl adenosine 3’,5’-cyclic monophosphate) and db-cGMP (N*, 02dibutyryl guanosine 3’.5’-cyclic monophosphate) (Sigma) at final concentrations ranging from 5X lo-” M to 1OF M were dissolved in LPS agar (LPS buffer plus 1.5% agar) and poured into 60x15 mm plastic Petri dishes. Vegetative amebae were harvested and the bacteria removed as described previously. lo7 amebae were spread onto each plate and on control plates containing no added cyclic nucleotide. The plates were incubated at 22°C in the dark and their development followed until fruiting bodies formed on the control plates (30 h).

RESULTS Extracellular development

3

!

6

9 \

12

15 \

16

21 \

Fi,o. 1. Ahscissu: time after removal of bacteria and develonmental mornholoav (hours): ordirroie: nmol CAMP or cGMP/lOs amebae. Extracellular cAMP and cGMP during develonment of P. violaceum. Vegetative P. violaceum were put on drums after removing the bacteria (T,). At 14 h intervals the drums were shifted into fresh chambers containing 22 ml of buffer and samples of the old buffer were frozen at -20°C. The frozen samples were thawed and assayed for CAMP (e) or cGMP 10) as described in Materials and Methods. &

-,\

II

CAMP and cGMP during of P. violaceum

Although P. violaceum does not use CAMP as its acrasin, it does excrete it [l]. The experiment illustrated in fig. 1 shows the levels and time course for CAMP excretion in this species. In this experiment wildtype amebae were allowed to develop on the surface of a dialysis membrane (as described under Materials and Methods). Most of the CAMP excreted will dialyze into the buffer below the membrane and therefore be protected from possible hydrolysis by phosphodiesterases. CAMP was excreted early in development with a peak rate of 12-13 pmol/90 min/lOs cells at the time aggregation centers were first visible. The amount excreted fell off rapidly as development proceeded, reaching a plateau value of 2-3 pmol cAMP/90 min/ 10” amebae throughout the remainder of development. In several experiments the absolute level of CAMP excreted varied in amount from

g-30 pmol/9O min/lO* amebae but the time course always remained the same. In this experiment not all of the bacteria were washed away when the amebae were deposited on the dialysis membrane. Control experiments using only E. coli B/r plated on the surface of the dialysis membrane showed little or no excretion of CAMP by the bacteria (data not shown): mdicating that the extracellular CAMP assayed in our experiments was derived from the amebae. Recently, Wurster et al. [lg] have shown that P. violaceum synthesized cGMF in response to a pulse of the P. V~&CWB attractant. No attempt was made to distinguish between extra- and intracelluku cGMP in these experiments. In order to determine the location of the cGMP synthesized, the samples assayed for extracellular CAMP levels (shown in fig. 1) were also assayed for extracellular GGMP levels.

268

Hanna, Klein and Cox

3

6

9

12

15

18

21

1

24

\ 1 I ~!~~;~,@? s&g /

4

8

12

16

20

24

Fig. 3. Abscissa: time after removal of bacteria and developmental morphology (hours); ordinate: pmol cAMP or cGMP hydrolyzed/mg protein/min. Cell-associated CAMP and cGMP phosphodiesterase activity during development of P. violaceum. Vegetative P. violaceum were filtered onto 0.45 pm Millipore filters and allowed to develop as in fig. 2. At la h intervals amebae were resuspended from the filters, pelleted by centrifugation and frozen at -20°C. The thawed pellets were assayed for phosphodiesterase activity and protein concentration as described in Materials and Methods. 0, CAMP phosphodiesterase activity; 0, cGMP phosphodiesterase activity.

I i d&y+,;.

1 +a&

i Fig. 2. Abscissa: time after removal of bacteria and developmental morphology (hours); ordinate: (A) pmol cAMP/108 amebae; (B) pmol cGMP/lO* amebae. Total CAMP and cGMP during development of P. violaceum. Vegetative P. violaceum, free of bacteria, were filtered onto 0.45 pm Millipore filters. The filters were placed on buffer saturated absorbent pads. At 1 h intervals, filters were inverted in 5% TCA and the cyclic nucleotides extracted and assayed as described in Materials and Methods. The data are corrected for recoveries of cyclic nucleotide and expressed as pmol/lO* amebae. (A) CAMP, (B) cGMP.

Very little cGMP was detected in this or subsequent experiments (less than l-2 pmol/90 min/ lo8 amebae). Total CAMP and cGMP during development Amebae were dispensed onto Millipore filters and allowed to develop. At 1 h interExp Cell Res 122 (1979)

vals the cyclic nucleotides were extracted from the amebae and the filter. Millipore falters have been shown to retain most of the extracellular CAMP [28], and thus extraction of the filter containing the amebae gives an accurate measure of the total cyclic nucleotide. This was verified in our original experiments by extracting the pads under the Millipore filter. No CAMP was detected in the assays of the pad extract. As shown in fig. 2A the amount of CAMP synthesized by P. violaceum increased more than 15-fold throughout development, with the sharpest increase in total CAMP occurring at the end of aggregation and continuing through culmination (15-25 h after removal of bacteria) . During early development the pattern of CAMP synthesis was not consistent. In two other experiments the

CAMP and cGMP modulation of development in P. violaceum apparent 4 h periodicity shown in fig. 2A was not observed. However, an overall increase in cAMP always occurred during development, with the greatest increase at the end of aggregation and continuing through culmination. The total amount of cGMP synthesized during development is shown in fig. 2B. At early stages of development very little cGMP was synthesized. Highest levels of cGMP were observed when cells were actively streaming. In this experiment 3 peak concentrations of cGMP were recorded during the period of development from mid-aggregation until the end of aggregation. In two other experiments only 2 peaks were observed during the same developmental time. The synthesis of cGMP decreased rapidly at the end of aggregation, reaching a basal level of approx. 2 pmol/h1108 amebae. Effect of excess extracellular cyclic nucleotides on development 0s P. violaceum LPS agar plates containing either CAMP, cGMP or their dibutyryl derivatives at concentrations from 5~ lo-” to 1OV M were spread with vegetative amebae that had been washed free of bacteria. The development of the amebae in the presence of added cyclic nucleotide was followed and compared with control cells developing in the absence of cyclic nucleotides. No significant difference in development was observed in the presence of either CAMP or cGMP up to 5x lop3 M except for a slightly earlier time of aggregation in the presence of either cyclic nucleotide at its highest concentration. Fruiting bodies were formed at approximately the same time on the plates 66th and without cyclic nucleotides. A similar result has been obtained by

269

George [9]. The inability of cyclic nucleotides to affect development in P. ~~0~~~~~~ is in contrast to its effect on devel in P. pallidzm [8, 93 another s slime mold not using cyclic nucleotides as a chemoattractant. Cell-associated cyclic nucleotide phosphodiesterase in P. violaceum Both Bonner et al. [I] and Gerisch et al. [2] have shown that p3. violacezun produces an extracellular phosphodiesterase. This phosphodiesterase is expressed rnax~rna~~~ at the end of the growth phase and tbro~~h early development [2]. As shown in fig. 3, P. violaceum has a cell-associated cyclic nucleotide phosphodiesterase in addition to the extracellular enzyme. The se& associated hydrolytic activity may be to intracellular and/or membrane-be enzyme(s). At the onset of sta~v~t~~~, the cell-associated CAMP phos~hod~e$~erase activity was at its highest. The activity dropped rapidly and was followed second peak of activity beginning at aggregation. The activity th later stages of development re than that of aggregation phase cells. Control experiments using E. coli above showed no cyclic n phodiesterase activity (data not shown), indicating that the phosphodiesterase was derived from the amebae and not from contaminating bacteria. When the samples phesfrom fig. 3 were assayed for c phodiesterase, the pattern was very similar to the cAMP phosphodiesterase pattern At the moment we do not know if because there is a single enzyme responsible for both hydrolytic activities or if two or more different enzymes are c~ordi~~~e~~ regulated. Preliminary experiments suggest either that there is more than one, or that the substrate specificity of the enzyme

270

Hanna, Klein and Cox

initially present is altered with time (Hanna & Cox, unpublished). DISCUSSION These results, with those of others, make it clear that during P. violaceum development there is an initial excretion of CAMP [this paper; 11.Following the excretion of CAMP and beginning in mid-aggregation, and ending prior to culmination, cGMP is synthesized and accumulated but not excreted to any detectable level (this paper). Cellassociated phosphodiesterase activity falls from an initial high level when the food source is removed, and rises again at about the time cGMP is synthesized, only to fall again during late culmination (this paper). An extracellular phosphodiesterase is also present during the transition from growth to starvation [ 1, 21. Shortly after cGMP accumulation falls off cellular CAMP levels begin to increase reaching a concentration at the fruiting body stage roughly four times that present at mid culmination (this paper). Wurster et al. [18] have shown that aggregation competent cells suspended in buffer respond to a pulse of the crude P. violaceum acrasin (or chemoattractant) by synthesizing cGMP. Our finding that cGMP levels rise at about the time cells begin to stream toward aggregation centers (8 h, fig. 2) agrees with their observations, although the amount of accumulation we observe is several-fold less than that observed by Wurster et al., perhaps because their data comes from experiments with cells in suspension. In addition, P. Pan (in preparation) has shown that most of the cGMP detected by fluorescent tagged antibodies is in the nucleus at these same times. CAMP begins to accumulate intracellularly at the end of aggregation in P. violaceum and continues to do so throughout culminaExp Cell Res 122 (I 979)

tion and fruiting body development. Experiments by Hohl et al. [8] and George [9] in a related species, P. pallidum, show that high concentrations of CAMP and cGMP added to developing amebae interfere with normal development and induce stalk cell formation. High levels of CAMP, cGMP and their dibutyryl derivatives do not have similar effects on P. violaceum, as reported here and by George [9]. We have shown that CAMP accumulation at later stages of development is intracellular, and thus for extracellular cyclic nucleotides to affect development they must be taken up by the amebae. The use of high concentrations of CAMP to affect development in P. pallidurn suggests that this uptake by the amebae is not very efficient. Unlike P. pallidurn, P. violaceum produces and excretes an extracellular phosphodiesterase [ 1, 21 and also has a cell associated phosphodiesterase (this paper). The combined action of both phosphodiesterases in P. violaceum may effectively lower the CAMP concentration to the point where it can not be taken up by the amebae. To show a significant effect -of external CAMP or cGMP on stalk cell formation in P. violaceum it may be necessary therefore to use P. violaceum mutants [29] defective in phosphodiesterase, as has been done in D. discoideum [6], or to inhibit the phosphodiesterase activity using one of many possible inhibitors [30]. Goldberg and collaborators [31, 321 have suggested that the ratio of cAMP/cGMP may be the critical variable in determining which pathways a cell will take during development. These authors have shown that CAMP and cGMP levels have opposing affects on cellular events as diverse as contractility of rat myocardial muscle, mitogenic stimulation of lymphocytes, and fibroblast proliferation. This communica-

CAMP and cGMP modulation

tion shows that the levels of CAMP and cGMP change as a function of developmental stage and that the pattern of change is different for CAMP and cGMP. It seems possible that P. violaceum may provide a suitable system for determining whether or not the cAMP/cGMP ratio is important in controlling developmental processes. This study was supported by funds from Research Grant No. AI-09392 of the NIH. We also benefited from the central equipment facilities in the Biology Department, Princeton University, supported by the Whitehall Foundation. M. H. was a postdoctoral trainee of the NCI, grant no. ST32CA 09167-02.

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13. - Biochem biophys res commun 79 (1977) 611. 14. Sussman. M & Schindler. J. Differentiation 10 ’ ’ (1978) 1.’ 15. Mato, J M & Koniljn, T M, Development and differentiation in the cellular slime molds (ed P Cappuccinelli & J M Ashworth) p. 93. Elsevieri North-Holland Biomedical Press, Amsterdam (1977). 16. Mato, J M, Kreus, F A, van Haastert, P J M & Konijn, T M, Proc natl acad sci US 74 (1977) 2348. 17. Wurster, B, Schubiger, K, Wick, V & Gerisch, 6; FEBS lett 76 (1977) 141. 18. Wurster, B, Bozzaro, S & Gerisch, 6, Cell biol int reports 2 (1978) 61. 19. Vogel, H J & Bonner, D M, J biol &em 218 (1956) 97. 20. Bonner, J T, J exp zoo1 IO6 (1947) 1. 21. Newell, P C, Telser, A & Sussman, M, J bacteriol 100 (1969) 763. 22. Bonner, J T, Barkley, D S, Hall, E M, Konijn, T M, Mason, J W, O’Keefe III, G & Wolfe, P B, Dev biol20 (1969) 72. 23. Steiner, A L, Parker, C W & Kipnis, D M: J biol them 247 (1972) 1106. 24. Hanna, M H & Cox, E C, Dev bio162 (1978) 206. 25. Brooker, G, Thomas, L J Jr & Appleman, M M, Biochemistry 12 (1968) 4177. 26. Klein, C, J biol them 250 (1975) 7134. 27. Lowry, 0 H, Rosebrough, N J, Farr, A L &. Randall, R J, J biol them 193 (1951) 265. 28. Brenner, M, Dev bio164 (1978) 210. 29. Warren, A 3, Warren, W K & Cox, E C, Genetics 83 (1976) 25. 30. Chasin, M & Harris, D, Adv cyclic nucleic res 7 (1976) 225. 31. Goldberg, N D, Haddox, M K, Dunham, E. Lopez, C & Hadden, J W, Control of proliferation in animal cells (ed B Clarkson & R Baserga) p. 609. Cold Spring Harbor laboratory, New York (1974). 32. Goldberg, N D, Haddox, M K, Nicol, S E, Glass, D B, Sanford, C H, Kuehl, F A Jr & Esteusen, R, Adv cyclic nucleotide res 5 (1975) 307. Received February 13, 1979 Accepted March 26, 1979

Exp Cell Res 122 (1979)