Experimental Cell Research 95 (1975) 79-87
LOCALIZATION
OF ADENYLATE
DICTYOSTELIUM
CYCLASE
IN
DZSCOZDEUM
II. Cytochemical Studies on Whole Cells and Isolated Plasma Membrane Vesicles L. S. CUTLER and E. F. ROSSOMANDO Department
of Oral
Biology, The University of Connecticut Farmington, CT 06032, USA
Health Center,
SUMMARY Cytochemical procedures for the localization of adenylate cyclase and 5’-nucleotidase were performed on stationary phase cultures of axenically grown Dictyostelium discoideum. To confirm the cytochemical observations made in vivo, similar studies were done on an isolated plasma membrane vesicle fraction known to contain both enzyme activities. In vivo, reaction product indicative of adenylate cyclase activity was seen only at the intracellular aspect of the plasma membrane while 5’-nucleotidase reaction product was only seen at the cell membrane predominantly in association with the extracellular aspect of the membrane. The polarization of reaction products on opposite sides of the plasma membrane was also observed in the isolated membrane vesicle fraction indicating that the membrane vesicles formed by amphotericin B lysis of these cells are derived from the plasma membrane. However, the membrane vesicles appeared inverted (inside-out) by the cytochemical observations. The cytochemical localization of these enzymes in plasma membrane vesicles both complements and confirms the observations made in vivo. Based on previous biochemical data and on the present cytochemical observations, it is concluded that the probable active site of adenylate cyclase faces the intracellular aspect of the plasma membrane and the probable active site of 5’-nucleotidase faces the extracellular surface of the plasma membrane.
Localization of adenylate cyclase at the plasma membrane enables the enzyme to act as the transducer between external stimuli and the intracellular effector, cyclic adenosine3’,j’-monophosphate (CAMP). From biochemical data [2, 11, 193 it has been suggested that the active site of adenylate cyclase is located on the intracellular aspect of the plasma membrane. The current investigation was undertaken to cytochemically localize the site of adenylate cyclase activity in the eucaryotic organism Dictyostelium discoideum, in 6-751813
which CAMP can apparently act as both a ‘first’ and ‘second’ messenger [ 1, 71. In order to establish the validity of the cytochemical localization of adenylate cyclase, cytochemical studies of S-nucleotidase localization were done. 5’-Nucleotidase has been confirmed as an ‘ectoenzyme’ (an enzyme associated with extracellular aspect of the plasma membrane) in mammalian cells [3, 4, 193 and in glutaraldehyde-fixed D. discoideum cells [9]. In an effort to confirm the cytochemical localization of adenylate cyclase and S-nucleotidase in ExptlCell
Res 95 (1975)
80
Cutler and Rossomundo
the intact cells, similar cytochemical studies were done on an isolated plasma membrane vesicle fraction known to contain both enzyme activities [22].
0.05 M cacodylate buffer (pH 7.2) followed by two washes in 0.1 M Sorenson’s phosphate buffer (PH 7.2) at 4°C. The cells were post-fixed for I h in 1% osmium tetroxide and processed and sectioned as described above. Adenylate cyclase olasma membrane
MATERIALS
AND METHODS
and 5’-AMPasr localization vesicles. A purified vesicular
it1
prep‘aration of plasma membrane was obtained by the-lysis of stationary phase cells of axenic strain AX-3 of D. disroideum with amnhotericin B. The membrane fractions were characterized biochemically and morphologically to confirm their origin [22]. The vesicle fraction was fixed in 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2) for 30 min at 4°C and cytochemically assayed for adenylate cyclase and 5’-nucleotidase activity as described above and then routinely processed and embedded. After fixation as described above, analysis of adenylate cyclase activity and 5’-nucleotidase activity was measured in whole cells and plasma membrane fractions using procedures described previously [22]. A similar analysis was done following fixation and subseouent incubation in 4 mM lead mtrate for 30 min at 37dC. Fixation procedures caused a 75-80% reduction in adenylate cyclase activity and a 60-65 % reduction in 5’-nucleotidase activity. No further reduction in either 5’nucleotidase or adenylate cyclase activity was seen after fixation and incubation in 4 mM lead nitrate.
cyclase localization in viva. Early stationarv chase cultures of the axenic strain (AX-3) of D. discoidearn [ 161 were fixed for 3 h in 2.5 % glutaraldehvde in 0.05 M cacodvlate buffer (oH 7.2) at 4°C. This fixation procedure was adopted after several trial experiments because it gave a diminished labelling reaction which facilitated a more discreet localization of reaction product. Following fixation the cells were concentrated by centrifttgation (5 000 g for 10 min) and washed for 18 h in 0.05 M cacodvlate buffer containing 7% (w/v) sucrose added. The cells were then resuspended in 3.0 ml of a modified Howell & Whitfield [6] incubation media composed of 80 mM Tris-maleate buffer (pH 7.3) containing 8% sucrose, 0.5 mM 5’-adenylyl-imidodiphosphate (AMP-PNP), 4 mM lead nitrate, 2 mM magnesium sulfate, 40 mM sodium fluoride and 50 mM dithiothreitol (an effective blocker of CAMP phosphodiesterase in this organism [ 131). The addition of sodium fluoride and dithiothreitol to the media inhibits ATPases and CAMP phosphodiesterase which could act as sources of phosphate that could Fig. 1. Lower power picture showing an axenically lead to spurious staining reactions. Control samples grown cell of the strain AX-3 of D. discoideum. Note were treated as above but were either incubated in that this particular cell is multinucleated (N). Several media with 4 mM ATP substituted for the substrate large and small vesicles (large arrows) and mito(AMP-PNP), in media without substrate or were boiled chondria (m) are seen in the central cytoplasm while for 5 min just before incubation in complete reaction some subsurface vesicles (small arrows) and glycogen media. All samples were incubated for 45 mitt at 30°C (g) are seen peripherally. A few strands of endoplasmic with gentle agitation. The incubation media was then reticulum (er), can also be seen. Short pseudopods removed and the cells washed twice with cold (4°C) project from cell surface. Section stained with 0.05 M cacodylate buffer (pH 7.2) followed by two uranyl acetate the and lead citrate. (X 12500). washes in 0.1 M Sorenson’s phosphate buffer (pH 7.2). Fig. 2. Low power picture showing a D. discoideum The cells were post-fixed for 1 h in 1% phosphate cell after incubation for adenylate cyclase activity. A buffered osmium tetroxide and then dehydrated bulbous pseudopod (large arrow) projects from the cell through a graded series of ethanols, propylene oxide surface. Small deposits of adenylate cyclase reaction and embedded in Epon 812 [8]. Control experiments product can be seen along the cell surface (small arhave shown that post-fixation in phosphate buffer does The nucleus (N), mitochondria (m), internal not give inappropriate lead precipitation but does yield rows). vesicles and cytoplasm are free of reaction product. better fixation. Thin sections (600-900 A) were cut on Section stained with uranyl acetate and lead citrate. LKB ultratome III, stained with methanolic uranyl (X 17000). acetate [ 183 alone or doubled stained with methanolic Figs 3 and 4. High magnification micrographs showing uranyl acetate and lead citrate [20] and examined on a localization of adenylate cyclase reaction product (arZeiss EM 10 electron microscope. rows) in association with the intracellular aspect of the plasma membrane of D. discoideum. (Fig. 3, x 200 000; 5’-Nucleotidase (5’-AMPase) localization in viva. Cells were fixed and washed as described above. The fig. 4, x250000). cells were then resuspended and incubated in 3.0 ml of Fig. 5. Low power micrograph showing reaction product of 5’-AMPase (arrows) at the plasma membrane media containing 80 mM Tris-maleate-sucrose buffer of a liquid culture grown cell (0. discoideum). (pH 7.3), 1 mM 5’-AMP, 3 mM lead nitrate and 2 mM magnesium sulfate for 30 min at 37°C. Control cells ‘(X40000). were incubated in media without substrate or in media Fig. 6. High power micrograph showing the reaction product of 5’-AMPase in association with the extracelwith P-glycerophosphate substituted for the 5’-AMP. Additional controls were incubated in whole media lular surface of the plasma membrane of the cell shown in fig. 5. A small amount of reaction product seems to after boiling for 5 min or in media containing 10 mM sodium fluoride [4]. The reaction was terminated by be associated with the inner aspect of the cell removing the incubation media and washing twice in membrane. (x250000).
Adenylate
Exptl Cell Res 95 (I 975)
Localization
of adenylate
cyclase in D. discoideum.
Exptl Cell
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81
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82
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RESULTS Structural features of stationary phase strain AX-3 D. discoideum grown in liquid culture
The cells are essentially spherical in shape with short pseudopodia projecting from their surface. The plasma membrane is distinct and appears as a well demarcated trilaminar structure. The cytoplasm of the cells contains numerous vesicles of varying size, rod to oval shaped mitochondria and inconspicuous Golgi units all in a juxtanuclear position. The cytoplasm also contains numerous polyribosomes, a few short strands of rough endoplasmic reticulum (ER), peripherally situated pools of glycogen, and several subsurface vesicles. At times the organism appears to be multinucleated under the conditions of this study (figs 1, 2). Localization of adenylate cyclase in vivo
Reaction product indicative of adenylate cyclase activity was seen along the plasma membrane. Significant reaction product was not observed in association with the membranes of the intracellular vacuoles (fig. 2). Reaction product was not found in the nuclei, mitochondria, rough ER, Golgi, or free in the cytosol. At high magnification the reaction product was observed in close association with the intracellular aspect of the plasma membrane (figs3, 4j. Reaction product was not observed at the outer (extracellular) surface of the plasma membrane. Localization (5’-AMPase)
of 5’-nucleotidase in vivo
S-Nucleotidase (S-AMPase) reaction product was seen only at the plasma membrane (fig. 5). The quantity of reaction product Expfl
Cell
Res 95 (197.5)
seen at the surface of different cells in the different specimens showed a moderate degree of variability with some cells demonstrating good stainability while others were completely negative. Higher resolution examination showed that the reaction product was localized predominantly on the outer or extracellular surface of the plasma membrane (fig. 6). Localization of 5’-AMPase and adenylate cyclase activity in plasma membrane vesicular fraction
The detailed morphologic and biochemical characteristics of this membrane fraction have been described elsewhere [22]. Adenylate cyclase reaction product was generally observed in association with the outer surface of the vesicle membrane (fig. 7). Only occasional vesicles showed reaction product along the inner surface of the limiting membranes. In contrast, reaction product for S-AMPase was seen in association with the inner surface of the vesicle membrane and there was accumulation of reaction product in many vesicles (fig. 8). Some vesicles had reaction product associated with their external surface but these were rarely seen. Fig. 7. Micrograph of a purified plasma membrane vesicle fraction from D. discoideum following incubation to demonstrate adenylate cyclase. Note that the reaction product is associated with several vesicles (large arrows) and membrane fragments. Inset: Higher power micrograph showing a vesicle from the above preparation. Note that the adenylate cyclase reaction product is associated with the outer surface of the vesicle (nrrows). Section stained with uranyl acetate and lead citrate. (X 125OOO;inser, X200000). Fig. 8. Micrograph of a purified plasma membrane vesicle fraction from D. discoideum after incubation for 5’-AMPase activity. Reaction product is seen inside the majority of vesicles (large arrows). An occasional vesicle appears to have reaction product associated with its outer surface (small arrows). Inset: High power micrograph of a vesicle from the above preparation showing 5’-AMPase reaction product associated with the inner aspect of the vesicle membrane (arrows).
(X50000;
inset,
X 200000).
Localization
of adenylate cyclase in D. discoideum.
ExptlCell Res
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83
95 (1975)
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Cutler and Rossomando
Localization
of adenylate
Controls
In substrate deleted, inhibited or boiled controls, in vivo and vesicles, there was little or no reaction product observed. In the S-nucleotidase experiments there were small amounts of reaction product seen in the P-glycerophosphate and sodium fluoride inhibited controls. Such minute quantities of reaction product probably represent incomplete inactivation of S-AMPase by sodium fluoride and slight reactivity of non-specific phosphatases in the case of the P-glycerophosphate. There was a completely different pattern of reaction product deposition in whole cells incubated with ATP substituted for AMP-PNP in the adenylate cyclase experiments. Reaction product was seen in association with both the internal and external aspects of the plasma membrane and in the intracristal spaces of the mitochondria (figs 9, 10). Reaction product was not seen in association with the internal vesicular system of the cells (fig. 9).
DISCUSSION The results of the current study indicate that reaction product indicative of the site of release of pyrophosphate by the action of adenylate cyclase on an ATP analog (AMP-PNP) is cytochemically localized Fig. 9. Electron micrograph of D. discoideum cells following incubation in adenylate cyclase medium using ATP as a substrate. Reaction product is seen on both sides of the plasma membrane (arrows) and within the mitochondria (m). The intracellular vacuoles (v) do not contain reaction product. Unstained section (X 19000). Fig. IO. Higher magnification electron micrograuh showing themitochondria of a D. discoideum &I1 incubated for adenylate cyclase activity with ATP as a substrate. Note ihe discrete localiz&ion of reaction product within the mitochondria. The reaction product appears to be confined to the intracristal space. Unstained section (X 76 OW).
cyclase in D. discoideum.
ZZ
8.5
along the inner surface of the plasma membrane of stationary phase cultures of D. discoideum. The discrete localization of reaction product when AMP-PNP is used as a substrate compared with the more diffuse distribution of reaction product when ATP is used as a substrate tends to confirm the specificity of the staining reaction. The localization of adenylate cyclase reaction product at the inside of the plasma membrane and the absence of such reaction product on intracellular vesicles or other cellular organelles is also consistent with the distribution of CAMP observed by Pan et al. [12] using immunofluorescent techniques. The present cytochemical observations are also consistent with biochemical data from other systems [ 11, 191 which suggested that adenylate cyclase released CAMP at the intracellular surface of the plasma membrane. The localization of adenylate cyclase reaction product in the present study at the inner surface of the plasma membrane differs from the localization of this reaction product reported by Reik et al. [15] and Howell & Whitfield [6] who studied rat liver or rat islets of Langerhans. These investigators localized adenylate cyclase reaction product on the outer surface of the plasma membrane of the cells. There are two likely explanations for this variance in results. First, the reaction product obtained in the cytochemical studies on adenylate cyclase may always appear on the side of the plasma membrane opposite the biochemically defined active site. If this were true, adenylate cyclase would be situated on the external surface of stationary phase D. discoideum cells. Such an observation would be appealing since these cells ‘secrete’ CAMP into the media during chemotaxis and aggregation [7]. However, immunofluorescent studies of stationary phase Exptl
Cell Res 95 (1975)
86
Cutler und Rossomando
D. discoideum cells by Pan et al. [ 121 suggest that such an interpretation would be incorrect. A second explanation of the observed differences in adenylate cyclase reaction product localization involves consideration of differences in the permeability of the plasma membranes of mammalian cells and of suspension grown D. discoideum cells. The differences in reaction product localization between the mammalian and the slime mold cells may reflect the ability of the substrate to completely penetrate the plasma membrane. Similar observations have been made for other cytochemical procedures [ 231. The difficulties involved in inferring enzyme active site localization from cytochemical observations of lead-phosphate reaction products are well known. However, in the current study two different enzymes, with distinctly separate biochemically localized active sites on the plasma membrane, were used as markers to help clarify such problems. The results of the current cytochemical studies on stationary phase D. discoideum are in good agreement with recent biochemical and cytochemical reports in both D. discoideum and other systems [3,4,9, 11, 14, 19,221. Interpretation of reaction product localization in the plasma membrane vesicle fraction is problematical. In direct contrast to the localization of the enzymes in vivo, the adenylate cyclase was observed on the external surface of the membrane vesicles while S-AMPase was localized on the internal surface of these vesicles. Several hypothesis can be derived to explain these observations but three propositions seem most appropriate. First, the enzymes may have shown a shift in their active site from one side of the membrane to the other. Such an event could occur if the enzymes Exptl
Cell
Res 95 (1975)
spanned the entire membrane [lo]. Such a shift seems unlikely since both enzymes showed a symmetrical change in location. A second alternative might be that the vesicles represent the end product of massive endocytosis caused by amphotericin B used in the lysis of the cells. Endocytic vesicles would have 5’-AMPase on their inner surface and adenylate cyclase on their outer surface. In this regard, 5’AMPase has been observed in association with the inner aspect of specific components of isolated Golgi fractions from rat liver [4]. Farquhar et al, [4] have pointed out that in this case the inner surface of secretory droplets of the Golgi fraction would become the outer surface of the plasma membrane during secretory discharge. Endocytosis is the reverse process of secretory granule discharge but the vesicles of both processes would have the same membrane configuration. The third mechanism by which inverted vesicles could be produced is the formation of vesicles from fragments of plasma membrane by a process which causes the membranes to vesicularize with their inner surface facing out. Plasma membrane vesicles of this configuration have been produced by a variety of methods from bacteria [5], human red blood cells [ 171 and murine plasmacytoma cells [21]. It is felt that this latter hypothesis is the most probable explanation for the inverted vesicles observed in the present study. Based on previously reported biochemical data and on the present cytochemical observations, it is concluded that the probable active site of adenylate cyclase in axenically grown, stationary phase D. discoideum cells faces the intracellular aspect of the plasma membrane, and the probable active site of 5’-nucleotidase faces the extracellular surface of the plasma membrane.
Localization
of adenylate
Acknowledgement and thanks are proferred to Mrs Connie Christian, Mrs Ann Hesla and Mr Charles Vaccaro for their technical help, to MS Carol Kowalczyk for her secretarial assistance and to Dr E. Golub and Dr G. Rodan for their helpful discussions during the course of this project.
REFERENCES 1. Bonner, J T, Proc natl acad sci US 65 (1970) 110. 2. Davoren, P R & Sutherland, E W, J biol them 238 (1963) 3016. 3. De Pierre, J W & Karnowsky, M L, Science 183 (1974) 1096. 4. Farquhar, M G, Bergeron, J J M & Palade, G E, J cell biol60 (1974) 8. 5. Futai, M, J memb biol 15 (1974) 15. 6. Howell. S L & Whitlield. M. J histochem cvtothem 20 (1972) 873. 7. Konijn, T M, Advances in cyclic nucleotide research (ed P Greengard, G A Robison & R Paoletti) vol. 1. Raven Press, New York (1972). Luft, J, J biophys biochem cytol 9 (l%l) 409. t : Malchow, D, Nagele, B, Schwarz, H & Gerisch, G, Em j biochem 28 (1972) 136. 10. Marchesi, V T, Hosp prac 8 (1973) 76. 11. @ye, I & Sutherland, E W, Biochim biophys acta 127 (1966) 347.
cyclase in D. discoideum.
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87
*12. Pan, P, Bonner, J T, Wedner, H J & Parker, C W, Proc natl acad sci US 71 (1974) 1623. 13. Pannbacker, R & Brevard, L, Science 175 (1972) 1014. 14. Pastan, I & Perlman, R L, Advances in cyclic nucleotide research (ed P Greengard, G A Robison & R Paoletti) vol. 1. Raven Press, New York (1972). 15. Reik, L, Petzold, G A, Higgins, J A, Greengard, P & Barnett, R J, Science 168 (1970) 382. 16. Rossomando, E F, Steffek, A J, Mujwid, D F & Alexander. S. EXD cell res 85 (1974) 73. 17. Steck, T L, Weinstein, R S, Strauss, H H & Wallath, D F H, Science 168 (1970) 255. 18. Stempak, J G & Ward, R T, J cell biol 22 (1964) 697. 19. Trams, E G & Lauter. C J. Biochim bionhvs _ _ acta 345 (1974) 180. 20. Venable, J H & Coggeshall, R, J cell biol25 (1965) 407. 21. Zachowski, A & Paraf, A, Biochem biophys res commun 57 (1974) 787. 22. Rossomandd, E F & Cutler, L S, Exp cell res 95 (1975) 67. 23. Ganote, C E, Rosenthal, A S, Moses, H L & Tice, L W, J histochem cytochem 17 (1969) 641. Received January 7, 1975 Revised version received February 19, 1975
Exptl
Cell
Res 95 (1975)
LOCALIZATION
OF ADENYLATE
DICTYOSTELIUM
CYCLASE
IN
DZSCOZDEUM
II. Cytochemical Studies on Whole Cells and Isolated Plasma Membrane Vesicles L. S. CUTLER and E. F. ROSSOMANDO Department
of Oral
Biology, The University of Connecticut Farmington, CT 06032, USA
Health Center,
SUMMARY Cytochemical procedures for the localization of adenylate cyclase and 5’-nucleotidase were performed on stationary phase cultures of axenically grown Dictyostelium discoideum. To confirm the cytochemical observations made in vivo, similar studies were done on an isolated plasma membrane vesicle fraction known to contain both enzyme activities. In vivo, reaction product indicative of adenylate cyclase activity was seen only at the intracellular aspect of the plasma membrane while 5’-nucleotidase reaction product was only seen at the cell membrane predominantly in association with the extracellular aspect of the membrane. The polarization of reaction products on opposite sides of the plasma membrane was also observed in the isolated membrane vesicle fraction indicating that the membrane vesicles formed by amphotericin B lysis of these cells are derived from the plasma membrane. However, the membrane vesicles appeared inverted (inside-out) by the cytochemical observations. The cytochemical localization of these enzymes in plasma membrane vesicles both complements and confirms the observations made in vivo. Based on previous biochemical data and on the present cytochemical observations, it is concluded that the probable active site of adenylate cyclase faces the intracellular aspect of the plasma membrane and the probable active site of 5’-nucleotidase faces the extracellular surface of the plasma membrane.
Localization of adenylate cyclase at the plasma membrane enables the enzyme to act as the transducer between external stimuli and the intracellular effector, cyclic adenosine3’,j’-monophosphate (CAMP). From biochemical data [2, 11, 193 it has been suggested that the active site of adenylate cyclase is located on the intracellular aspect of the plasma membrane. The current investigation was undertaken to cytochemically localize the site of adenylate cyclase activity in the eucaryotic organism Dictyostelium discoideum, in 6-751813
which CAMP can apparently act as both a ‘first’ and ‘second’ messenger [ 1, 71. In order to establish the validity of the cytochemical localization of adenylate cyclase, cytochemical studies of S-nucleotidase localization were done. 5’-Nucleotidase has been confirmed as an ‘ectoenzyme’ (an enzyme associated with extracellular aspect of the plasma membrane) in mammalian cells [3, 4, 193 and in glutaraldehyde-fixed D. discoideum cells [9]. In an effort to confirm the cytochemical localization of adenylate cyclase and S-nucleotidase in ExptlCell
Res 95 (1975)
80
Cutler and Rossomundo
the intact cells, similar cytochemical studies were done on an isolated plasma membrane vesicle fraction known to contain both enzyme activities [22].
0.05 M cacodylate buffer (pH 7.2) followed by two washes in 0.1 M Sorenson’s phosphate buffer (PH 7.2) at 4°C. The cells were post-fixed for I h in 1% osmium tetroxide and processed and sectioned as described above. Adenylate cyclase olasma membrane
MATERIALS
AND METHODS
and 5’-AMPasr localization vesicles. A purified vesicular
it1
prep‘aration of plasma membrane was obtained by the-lysis of stationary phase cells of axenic strain AX-3 of D. disroideum with amnhotericin B. The membrane fractions were characterized biochemically and morphologically to confirm their origin [22]. The vesicle fraction was fixed in 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2) for 30 min at 4°C and cytochemically assayed for adenylate cyclase and 5’-nucleotidase activity as described above and then routinely processed and embedded. After fixation as described above, analysis of adenylate cyclase activity and 5’-nucleotidase activity was measured in whole cells and plasma membrane fractions using procedures described previously [22]. A similar analysis was done following fixation and subseouent incubation in 4 mM lead mtrate for 30 min at 37dC. Fixation procedures caused a 75-80% reduction in adenylate cyclase activity and a 60-65 % reduction in 5’-nucleotidase activity. No further reduction in either 5’nucleotidase or adenylate cyclase activity was seen after fixation and incubation in 4 mM lead nitrate.
cyclase localization in viva. Early stationarv chase cultures of the axenic strain (AX-3) of D. discoidearn [ 161 were fixed for 3 h in 2.5 % glutaraldehvde in 0.05 M cacodvlate buffer (oH 7.2) at 4°C. This fixation procedure was adopted after several trial experiments because it gave a diminished labelling reaction which facilitated a more discreet localization of reaction product. Following fixation the cells were concentrated by centrifttgation (5 000 g for 10 min) and washed for 18 h in 0.05 M cacodvlate buffer containing 7% (w/v) sucrose added. The cells were then resuspended in 3.0 ml of a modified Howell & Whitfield [6] incubation media composed of 80 mM Tris-maleate buffer (pH 7.3) containing 8% sucrose, 0.5 mM 5’-adenylyl-imidodiphosphate (AMP-PNP), 4 mM lead nitrate, 2 mM magnesium sulfate, 40 mM sodium fluoride and 50 mM dithiothreitol (an effective blocker of CAMP phosphodiesterase in this organism [ 131). The addition of sodium fluoride and dithiothreitol to the media inhibits ATPases and CAMP phosphodiesterase which could act as sources of phosphate that could Fig. 1. Lower power picture showing an axenically lead to spurious staining reactions. Control samples grown cell of the strain AX-3 of D. discoideum. Note were treated as above but were either incubated in that this particular cell is multinucleated (N). Several media with 4 mM ATP substituted for the substrate large and small vesicles (large arrows) and mito(AMP-PNP), in media without substrate or were boiled chondria (m) are seen in the central cytoplasm while for 5 min just before incubation in complete reaction some subsurface vesicles (small arrows) and glycogen media. All samples were incubated for 45 mitt at 30°C (g) are seen peripherally. A few strands of endoplasmic with gentle agitation. The incubation media was then reticulum (er), can also be seen. Short pseudopods removed and the cells washed twice with cold (4°C) project from cell surface. Section stained with 0.05 M cacodylate buffer (pH 7.2) followed by two uranyl acetate the and lead citrate. (X 12500). washes in 0.1 M Sorenson’s phosphate buffer (pH 7.2). Fig. 2. Low power picture showing a D. discoideum The cells were post-fixed for 1 h in 1% phosphate cell after incubation for adenylate cyclase activity. A buffered osmium tetroxide and then dehydrated bulbous pseudopod (large arrow) projects from the cell through a graded series of ethanols, propylene oxide surface. Small deposits of adenylate cyclase reaction and embedded in Epon 812 [8]. Control experiments product can be seen along the cell surface (small arhave shown that post-fixation in phosphate buffer does The nucleus (N), mitochondria (m), internal not give inappropriate lead precipitation but does yield rows). vesicles and cytoplasm are free of reaction product. better fixation. Thin sections (600-900 A) were cut on Section stained with uranyl acetate and lead citrate. LKB ultratome III, stained with methanolic uranyl (X 17000). acetate [ 183 alone or doubled stained with methanolic Figs 3 and 4. High magnification micrographs showing uranyl acetate and lead citrate [20] and examined on a localization of adenylate cyclase reaction product (arZeiss EM 10 electron microscope. rows) in association with the intracellular aspect of the plasma membrane of D. discoideum. (Fig. 3, x 200 000; 5’-Nucleotidase (5’-AMPase) localization in viva. Cells were fixed and washed as described above. The fig. 4, x250000). cells were then resuspended and incubated in 3.0 ml of Fig. 5. Low power micrograph showing reaction product of 5’-AMPase (arrows) at the plasma membrane media containing 80 mM Tris-maleate-sucrose buffer of a liquid culture grown cell (0. discoideum). (pH 7.3), 1 mM 5’-AMP, 3 mM lead nitrate and 2 mM magnesium sulfate for 30 min at 37°C. Control cells ‘(X40000). were incubated in media without substrate or in media Fig. 6. High power micrograph showing the reaction product of 5’-AMPase in association with the extracelwith P-glycerophosphate substituted for the 5’-AMP. Additional controls were incubated in whole media lular surface of the plasma membrane of the cell shown in fig. 5. A small amount of reaction product seems to after boiling for 5 min or in media containing 10 mM sodium fluoride [4]. The reaction was terminated by be associated with the inner aspect of the cell removing the incubation media and washing twice in membrane. (x250000).
Adenylate
Exptl Cell Res 95 (I 975)
Localization
of adenylate
cyclase in D. discoideum.
Exptl Cell
ZZ
81
Res 95 (I 975)
82
Cutler and Rossomando
RESULTS Structural features of stationary phase strain AX-3 D. discoideum grown in liquid culture
The cells are essentially spherical in shape with short pseudopodia projecting from their surface. The plasma membrane is distinct and appears as a well demarcated trilaminar structure. The cytoplasm of the cells contains numerous vesicles of varying size, rod to oval shaped mitochondria and inconspicuous Golgi units all in a juxtanuclear position. The cytoplasm also contains numerous polyribosomes, a few short strands of rough endoplasmic reticulum (ER), peripherally situated pools of glycogen, and several subsurface vesicles. At times the organism appears to be multinucleated under the conditions of this study (figs 1, 2). Localization of adenylate cyclase in vivo
Reaction product indicative of adenylate cyclase activity was seen along the plasma membrane. Significant reaction product was not observed in association with the membranes of the intracellular vacuoles (fig. 2). Reaction product was not found in the nuclei, mitochondria, rough ER, Golgi, or free in the cytosol. At high magnification the reaction product was observed in close association with the intracellular aspect of the plasma membrane (figs3, 4j. Reaction product was not observed at the outer (extracellular) surface of the plasma membrane. Localization (5’-AMPase)
of 5’-nucleotidase in vivo
S-Nucleotidase (S-AMPase) reaction product was seen only at the plasma membrane (fig. 5). The quantity of reaction product Expfl
Cell
Res 95 (197.5)
seen at the surface of different cells in the different specimens showed a moderate degree of variability with some cells demonstrating good stainability while others were completely negative. Higher resolution examination showed that the reaction product was localized predominantly on the outer or extracellular surface of the plasma membrane (fig. 6). Localization of 5’-AMPase and adenylate cyclase activity in plasma membrane vesicular fraction
The detailed morphologic and biochemical characteristics of this membrane fraction have been described elsewhere [22]. Adenylate cyclase reaction product was generally observed in association with the outer surface of the vesicle membrane (fig. 7). Only occasional vesicles showed reaction product along the inner surface of the limiting membranes. In contrast, reaction product for S-AMPase was seen in association with the inner surface of the vesicle membrane and there was accumulation of reaction product in many vesicles (fig. 8). Some vesicles had reaction product associated with their external surface but these were rarely seen. Fig. 7. Micrograph of a purified plasma membrane vesicle fraction from D. discoideum following incubation to demonstrate adenylate cyclase. Note that the reaction product is associated with several vesicles (large arrows) and membrane fragments. Inset: Higher power micrograph showing a vesicle from the above preparation. Note that the adenylate cyclase reaction product is associated with the outer surface of the vesicle (nrrows). Section stained with uranyl acetate and lead citrate. (X 125OOO;inser, X200000). Fig. 8. Micrograph of a purified plasma membrane vesicle fraction from D. discoideum after incubation for 5’-AMPase activity. Reaction product is seen inside the majority of vesicles (large arrows). An occasional vesicle appears to have reaction product associated with its outer surface (small arrows). Inset: High power micrograph of a vesicle from the above preparation showing 5’-AMPase reaction product associated with the inner aspect of the vesicle membrane (arrows).
(X50000;
inset,
X 200000).
Localization
of adenylate cyclase in D. discoideum.
ExptlCell Res
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95 (1975)
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Cutler and Rossomando
Localization
of adenylate
Controls
In substrate deleted, inhibited or boiled controls, in vivo and vesicles, there was little or no reaction product observed. In the S-nucleotidase experiments there were small amounts of reaction product seen in the P-glycerophosphate and sodium fluoride inhibited controls. Such minute quantities of reaction product probably represent incomplete inactivation of S-AMPase by sodium fluoride and slight reactivity of non-specific phosphatases in the case of the P-glycerophosphate. There was a completely different pattern of reaction product deposition in whole cells incubated with ATP substituted for AMP-PNP in the adenylate cyclase experiments. Reaction product was seen in association with both the internal and external aspects of the plasma membrane and in the intracristal spaces of the mitochondria (figs 9, 10). Reaction product was not seen in association with the internal vesicular system of the cells (fig. 9).
DISCUSSION The results of the current study indicate that reaction product indicative of the site of release of pyrophosphate by the action of adenylate cyclase on an ATP analog (AMP-PNP) is cytochemically localized Fig. 9. Electron micrograph of D. discoideum cells following incubation in adenylate cyclase medium using ATP as a substrate. Reaction product is seen on both sides of the plasma membrane (arrows) and within the mitochondria (m). The intracellular vacuoles (v) do not contain reaction product. Unstained section (X 19000). Fig. IO. Higher magnification electron micrograuh showing themitochondria of a D. discoideum &I1 incubated for adenylate cyclase activity with ATP as a substrate. Note ihe discrete localiz&ion of reaction product within the mitochondria. The reaction product appears to be confined to the intracristal space. Unstained section (X 76 OW).
cyclase in D. discoideum.
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along the inner surface of the plasma membrane of stationary phase cultures of D. discoideum. The discrete localization of reaction product when AMP-PNP is used as a substrate compared with the more diffuse distribution of reaction product when ATP is used as a substrate tends to confirm the specificity of the staining reaction. The localization of adenylate cyclase reaction product at the inside of the plasma membrane and the absence of such reaction product on intracellular vesicles or other cellular organelles is also consistent with the distribution of CAMP observed by Pan et al. [12] using immunofluorescent techniques. The present cytochemical observations are also consistent with biochemical data from other systems [ 11, 191 which suggested that adenylate cyclase released CAMP at the intracellular surface of the plasma membrane. The localization of adenylate cyclase reaction product in the present study at the inner surface of the plasma membrane differs from the localization of this reaction product reported by Reik et al. [15] and Howell & Whitfield [6] who studied rat liver or rat islets of Langerhans. These investigators localized adenylate cyclase reaction product on the outer surface of the plasma membrane of the cells. There are two likely explanations for this variance in results. First, the reaction product obtained in the cytochemical studies on adenylate cyclase may always appear on the side of the plasma membrane opposite the biochemically defined active site. If this were true, adenylate cyclase would be situated on the external surface of stationary phase D. discoideum cells. Such an observation would be appealing since these cells ‘secrete’ CAMP into the media during chemotaxis and aggregation [7]. However, immunofluorescent studies of stationary phase Exptl
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D. discoideum cells by Pan et al. [ 121 suggest that such an interpretation would be incorrect. A second explanation of the observed differences in adenylate cyclase reaction product localization involves consideration of differences in the permeability of the plasma membranes of mammalian cells and of suspension grown D. discoideum cells. The differences in reaction product localization between the mammalian and the slime mold cells may reflect the ability of the substrate to completely penetrate the plasma membrane. Similar observations have been made for other cytochemical procedures [ 231. The difficulties involved in inferring enzyme active site localization from cytochemical observations of lead-phosphate reaction products are well known. However, in the current study two different enzymes, with distinctly separate biochemically localized active sites on the plasma membrane, were used as markers to help clarify such problems. The results of the current cytochemical studies on stationary phase D. discoideum are in good agreement with recent biochemical and cytochemical reports in both D. discoideum and other systems [3,4,9, 11, 14, 19,221. Interpretation of reaction product localization in the plasma membrane vesicle fraction is problematical. In direct contrast to the localization of the enzymes in vivo, the adenylate cyclase was observed on the external surface of the membrane vesicles while S-AMPase was localized on the internal surface of these vesicles. Several hypothesis can be derived to explain these observations but three propositions seem most appropriate. First, the enzymes may have shown a shift in their active site from one side of the membrane to the other. Such an event could occur if the enzymes Exptl
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spanned the entire membrane [lo]. Such a shift seems unlikely since both enzymes showed a symmetrical change in location. A second alternative might be that the vesicles represent the end product of massive endocytosis caused by amphotericin B used in the lysis of the cells. Endocytic vesicles would have 5’-AMPase on their inner surface and adenylate cyclase on their outer surface. In this regard, 5’AMPase has been observed in association with the inner aspect of specific components of isolated Golgi fractions from rat liver [4]. Farquhar et al, [4] have pointed out that in this case the inner surface of secretory droplets of the Golgi fraction would become the outer surface of the plasma membrane during secretory discharge. Endocytosis is the reverse process of secretory granule discharge but the vesicles of both processes would have the same membrane configuration. The third mechanism by which inverted vesicles could be produced is the formation of vesicles from fragments of plasma membrane by a process which causes the membranes to vesicularize with their inner surface facing out. Plasma membrane vesicles of this configuration have been produced by a variety of methods from bacteria [5], human red blood cells [ 171 and murine plasmacytoma cells [21]. It is felt that this latter hypothesis is the most probable explanation for the inverted vesicles observed in the present study. Based on previously reported biochemical data and on the present cytochemical observations, it is concluded that the probable active site of adenylate cyclase in axenically grown, stationary phase D. discoideum cells faces the intracellular aspect of the plasma membrane, and the probable active site of 5’-nucleotidase faces the extracellular surface of the plasma membrane.
Localization
of adenylate
Acknowledgement and thanks are proferred to Mrs Connie Christian, Mrs Ann Hesla and Mr Charles Vaccaro for their technical help, to MS Carol Kowalczyk for her secretarial assistance and to Dr E. Golub and Dr G. Rodan for their helpful discussions during the course of this project.
REFERENCES 1. Bonner, J T, Proc natl acad sci US 65 (1970) 110. 2. Davoren, P R & Sutherland, E W, J biol them 238 (1963) 3016. 3. De Pierre, J W & Karnowsky, M L, Science 183 (1974) 1096. 4. Farquhar, M G, Bergeron, J J M & Palade, G E, J cell biol60 (1974) 8. 5. Futai, M, J memb biol 15 (1974) 15. 6. Howell. S L & Whitlield. M. J histochem cvtothem 20 (1972) 873. 7. Konijn, T M, Advances in cyclic nucleotide research (ed P Greengard, G A Robison & R Paoletti) vol. 1. Raven Press, New York (1972). Luft, J, J biophys biochem cytol 9 (l%l) 409. t : Malchow, D, Nagele, B, Schwarz, H & Gerisch, G, Em j biochem 28 (1972) 136. 10. Marchesi, V T, Hosp prac 8 (1973) 76. 11. @ye, I & Sutherland, E W, Biochim biophys acta 127 (1966) 347.
cyclase in D. discoideum.
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*12. Pan, P, Bonner, J T, Wedner, H J & Parker, C W, Proc natl acad sci US 71 (1974) 1623. 13. Pannbacker, R & Brevard, L, Science 175 (1972) 1014. 14. Pastan, I & Perlman, R L, Advances in cyclic nucleotide research (ed P Greengard, G A Robison & R Paoletti) vol. 1. Raven Press, New York (1972). 15. Reik, L, Petzold, G A, Higgins, J A, Greengard, P & Barnett, R J, Science 168 (1970) 382. 16. Rossomando, E F, Steffek, A J, Mujwid, D F & Alexander. S. EXD cell res 85 (1974) 73. 17. Steck, T L, Weinstein, R S, Strauss, H H & Wallath, D F H, Science 168 (1970) 255. 18. Stempak, J G & Ward, R T, J cell biol 22 (1964) 697. 19. Trams, E G & Lauter. C J. Biochim bionhvs _ _ acta 345 (1974) 180. 20. Venable, J H & Coggeshall, R, J cell biol25 (1965) 407. 21. Zachowski, A & Paraf, A, Biochem biophys res commun 57 (1974) 787. 22. Rossomandd, E F & Cutler, L S, Exp cell res 95 (1975) 67. 23. Ganote, C E, Rosenthal, A S, Moses, H L & Tice, L W, J histochem cytochem 17 (1969) 641. Received January 7, 1975 Revised version received February 19, 1975
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