Localization of p-glycerophosphatase activity in the myxomycete Perichaena vermicularis

JAMES CRONSHAW AND IRIS CHARVAT Departtnerrt of Biological Sciences, University of California, Santa Barbara, California

Received June 12, 1972

CRONSHAW, J., and I. CHARVAT. 1973. Localization of (3-glycerophosphatase activity in the myxomycete Perichaena vermicr~laris. Can. J. Bot. 51: 97-101.

The myxomycete Perichaena verrnicrrlaris has been fixed with formaldehyde-glutaraldehyde at various stages of fruiting and the distribution of (3-glycerophosphatase determined at the electron microscope level using a modified Gomori procedure. Specimens from various stages of the developing fruiting body when incubated for 70-105 min at 39°C in a medium containing (3-glycerophosphate showed good localization of reaction product. High (3-glycerophosphatase activity was localized in single-membrane- bound vacuoles a t all stages of fruiting body formation. These vacuoles are similar to lysosomes of animals, higher plants, and fungi in that they are surrounded by a single membrane, contain cell com- ponents at various stages of disintegration, and have a high acid phosphatase activity.

Reaction product was also localized in the walls of mature spores and was variably associated with nuclei and mitochondria. Most probably the staining of some nuclei and mitochondria is a lead absorp- tion artifact.

CRONSHAW, J., et I. CHARVAT. 1973. Localization of (3-glycerophosphatase activity in the myxomycete Perichaena verrnicrrlaris. Can. J . Bot. 51 : 97-101.

Le myxomyctte Perichaena vermicularis a ete fix6 A la formaldehyde-glutaraldehyde a divers stades de la fructification, et la distribution de la p-glyctrophosphatase a kt6 dtterminee au microscope Clec- tronique a I'aide d'une modification de la mtthode Gomori. Chez des specimens a divers stades de dkveloppement de la fructification, incubes pendant 70-105 min a 39°C dans un milieu contenant du (3-glycerophosphate, il y avait une bonne localisation du produit de reaction. Une activite (3-glycero- phosphatasique &levee a Cte localisee dans des vacuoles limitees par une membrane simple a tous les stades de la formation de la fructification. Ces vacuoles sont semblables aux lysosomes des animaux, des plantes suptrieures et des champignons. Elles sont limit& par une membrane simple, elles con- tiennent des composantes cellulaires a divers stades de desintegration et elles ont une activite elevee de phosphatase acide.

Le produit de reaction a aussi ete IocalisC dans les parois des spores mCIres et Ctait invariablement associe aux noyaux et aux mitochondries. I1 est trks probable que la coloration de quelques noyaux et ~nitochondries est un artefact ~ C I a I'absorption du plomb. [Traduit par le journal]

Introduction

There are no reports in the literature of histo- chemical localization of acid phosphatase in the myxomycetes although acid phosphatase activity has been demonstrated biochemically in the plasmodia1 slime mold Physarum polycephalum (Hiittermann et al. 1970a) and histochemically in the food vacuoles of the cellular slime mold Dictyostelium discoidelrm (Gezelius 1970). Histo- chemical studies at the light and electron micro- scope levels have demonstrated acid phosphatase activity localized in vacuoles of several species of fungi (Gezelius 1970; Matile 1971; Pitt and Walker 1967; Pitt 1968; Wilson et al. 1970) and protozoa (see Aikawa and Thompson 1971; Bowers and Korn 1969; Esteve 1970). It is generally thought that these acid phosphatase containing vacuoles are a type of lysosome. Recently Matile and Weimken (1967) and Matile (1971) have isolated vacuoles from yeast and from the macroconidia of Nezrrospora crassa

that were a type of lysosome and contained high concentrations of a number of hydrolytic enzymes including acid phosphatase.

We have been investigating the fine structural changes that occur during the various stages of fruiting of the myxomycete Perichaena vermicu- laris and have observed vacuoles that morpho- logically appear to be a type of lysosome. The distribution of p-glycerophosphatase has been determined cytochemically in this organism using a modified Gomori procedure and it has been shown that the vacuoles that morpholog- ically resemble lysosomes are the major sites of p-glycerophosphatase activity.

Materials and Methods Fruiting bodies of Perichaetra vermicularis were crushed

in distilled water or 0.5% saline solution, and the spores were permitted to germinate. The resulting myxamoebae were mixed with a 24-h-old culture of E. coli, the mixture pipetted onto cornmeal agar plates and smeared with a flattened glass rod. These cultures were then grown in an

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98 CAN. I. BOT. VOL. 51, 1973

incubator under controlled conditions of temperature (26°C) and light cycle (8 h light and 12 h dark). After 7 to 12 days, fruiting bodies formed and the various stages of fruiting starting with the plasn~odium and continuing through spore formation were fixed for 2 h at room tem- perature using formaldehyde-glutaraldehyde in 0.1 M cacodylate buffer, pH 6.8. The material was then washed in tap water for about 39 h to remove the fixative.

P-Glycerophosphatase localization was carried out using a modified Gomori method (Dauwalder et a/ . 1969). The incubation medium consisted of lOml 0.05 M acetate buffer, pH 5.0; 13.25 mg Pb(N03)2; and 30.6 mg sodium P-glycerophosphate (Sigma Chemical Company). The medium was incubated for 12 h at 37°C and filtered before use. After the medium was filtered, the pH was readjusted to 5.0. Pieces of the plasmodium and the fruiting bodies were incubated in the medium with sub- strate for 70-105 min at 39°C. Controls were incubated in a similar medium without substrate. After incubation, the material was rinsed in 0.05y0 acetic acid for about 1 min, and postfixed in 2y0 cacodylate buffered osmium tetroxide for 2 h at room temperature. The samples were rinsed in buffer and then in water, dehydrated through a graded acetone series, and embedded in Epon.

The blocks were cut with a diamond knife on a Porter- Blum MT 1 ultramicrotome. Sections 1-2 micrometers (pm) thick were prepared for light microscope examina- tion and silver sections for electron microscopy. The sections were stained with uranyl acetate and Sato's (Sato 1968) lead stain or left unstained, and examined and photographed with a Siemens Elmiskop I.

Results In the development of the fruiting body of

Pericl~aena, the amoeba1 stage first fuses to give rise to zygotes. The zygotes divide and fuse to form a plasmodium which then forms localized initials that develop into plasmodiocarps. Within the plasmodiocarps cleavage of the protoplast takes place and spores are formed. A netlike capillitium is also formed in the plasmodiocarp so that the mature sporangium contains spores intermixed with capillitium filaments.

Specimens taken from the various stages of the developing fruiting body when incubated for 7@-105 min in the P-glycerophosphatase mixture showed good localization of reaction product. The amount of reaction product obtained using these incubation times was less in the spores once a thick wall had formed, most probably resulting from the difficulty in penetrating the spore wall.

In the plasmodium high activity was found in single-membrane-bound vacuoles (Fig. 1). These vacuoles varied in size and often contained recognizable Escherichia coli cells. Reaction product was also found associated with the

nuclear envelope, within the nucleus, and in some of the mitochondria (Fig. 1). In sections of the mitochondria the concentration of stain generally appeared as small circles.

When pieces of the plasmodium were incu- bated in a medium containing no substrate and examined unstained with the electron microscope only a small amount of deposited lead was ob- served, usually not associated with specific organelles (Fig. 2). In particular, the vacuoles that had heavy deposits of reaction product in the substrate-incubated material were devoid of stained contents in the no-substrate controls. When sections cut from the no-substrate con- trols were stained for electron microscopy with uranyl acetate and lead some structures within the vesicles were heavily stained, often semicircu- lar in outline (Fig. 3). There were also sometimes small amounts of stain associated with mito- chondria in these controls.

At the stage of development of the fruiting body when the protoplasm has cleaved to form young spores, large and small vacuoles are pres- ent. After incubation in medium containing P- glycerophosphate, heavy, often crystalline de- posits of reaction product were consistently present in these vacuoles (Fig. 4). Some of the mitochondria were also stained but the amount of stain associated with mitochondria was variable.

As the spores develop, a wall is formed and there is apparently fusion of vacuoles to form a large single-membrane-bound vacuole that con- tains clearly recognizable cytoplasmic compo- nents. Some of these components appear to be well preserved and others are at various stages of disintegration. In specimens incubated in reac- tion medium with substrate there is good local- ization of reaction product in these vacuoles (Fig. 5).

At maturity the spores have a comparatively thick wall and a large vacuole. The large vacuole shows good localization of reaction product but with the incubation times used the amount of stained material was much less than for the young spores (Fig. 6). The large vacuole also contained many fewer recognizable cytoplasmic structures. The spore wall showed some localiza- tion of reaction product. The lower quantity of reaction product in the vacuoles of spores with mature walls may be due to penetration prob-

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CRONSHAW AND CHARVAT: PERICHAENA VERMICULARIS 99

lems associated with the wall or may be due to a lower concentration of enzyme in the vacuole.

Discussion The stages of fruiting body formation exam-

ined for P-glycerophosphatase activity showed dense localizations of reaction product in various sizes in single-membrane-bound vesicles. In the plasmodium these vesicles contained cells of E. coli on which the organism was feeding but in the developing and mature spores the vacuoles with P-glycerophosphatase activity had intact cytoplasmic organelles and organelles at various stages of digestion. In addition to this vacuolar localization, the spore walls stained positive for P-glycerophosphatase and stain was observed associated with nuclei and mitochondria.

The existence of lysosomes in fungal cells has been demonstrated by cytochemical studies which have shown a articulate localization of acid phosphatase, and by biochemical studies which have shown that vacuoles can be isolated which contain several digestive enzymes in con- centrated form. At the light microscope level Zalokar (Zalokar 1960, 1965) demonstrated the presence of P-glycerophosphatase in Neurospora crassa. Pitt and Walker (1967) showed particu- late staining in several fungi using the Gomori and azo dye techniques. Pitt (1968) observed a particulate distribution of enzymes other than P-glycerophosphatase in Botrytis cinera. The particles stained positively for acid deoxyribo- nuclease I[, galactasidase, and a number of esterases. Several phytopathogenic fungi have also been shown to localize P-glycerophospha- tase, arylsulfatase, and deoxyribonuclease in cell particles (Armentrout et al. 1968; Arinentrout and Wilson 1969; Wilson et al. 1970).

At the electron microscope level, particulate localization of P-glycerophosphatase was demon- strated in the phytopathogens Ceratocystis fim- briata and C. fagacearunz (Wilson et al. 1970). The fungi were incubated in Gomori medium for 24 h at 37"C, however, and the prolonged incuba- tion period makes the results suspect. a-Glycero- phosphatase was found in the granules and cell wall of yeast cells (Gunther et al. 1967). When the cells were repressed, by placing them in a phosphate rich medium, there was a decrease in the precipitation of the enzyme. The cellular slime mold Dictyostelium discoideurn contains

food vacuoles positive for acid phosphatase (Gezelius 1970). All substrates used including P-glycerophosphate, p-nitrophenyl phosphate, and glucose-6-glycerophosphate resulted in iden- tification of the reaction product in the digestive vacuoles.

Vacuoles of fungi, surrounded by a single membrane, have been shown biochemically to contain lytic enzymes. Isolated preparations of purified yeast vacuoles were found to have 20- to 30-fold specific activity of acid hydrolases compared with activities in the lysed protoplasts (Matile and Wiemken 1967). Matile (1969) con- cludes that yeast vacuoles are apparently typical lysosomes having characteristics of animal cell lysosomes. Matile (1971) later isolated vacuoles containing high concentrations of a number of hydrolytic enzymes from macroconidia of Neuro- spora crassa.

Narkates et al. (1968) found a correlation between the biochemical 'and cytochemical acid phosphatase activity with phases of growth in Candida albicans. During the logarithmic growth, there is a linear increase of the enzyme activity with the age of the cells during the first 14 h ; the cytochemical distribution of stain in the cyto- plasmic granules and vacuoles correlated with the change in intensity of the biochemical results.

'The activity of acid phosphatase has been examined biochemically in the plasmodia1 slime mold Plzysarum polycephalum. The assay showed a continuous increase in enzymic activity during the mitotic cycle of the plasmodium (Hiittermann et al. 1970a). During spherulation the increase in specific acid phosphatase activity was about two- fold (Huttermann et al. 19706).

These observations on hydrolytic enzyme ac- tivity and the demonstrations of lytic enzymes in single-membrane-bound vacuoles together with the results presented in the present paper support the view that lysosomes are present in fungi and slime molds. The vesicles present in the stages of fruiting body formation of Peri- chaena vermicularis concentrate P-glycerophos- phatase, are surrounded by a single membrane, and contain cellular material in various stages of disintegration. They are thus comparable to the lysosomes of animals, higher plants, and fungi.

There are several reports of P-glycerophospha- tase localization in plant cell walls (Hall 1969; Halperin 1969 ; Knox and Heslop-Harrison

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100 CAN. J BOT. VOL. 51, 1973

1969; Poux 1970). In yeast and Neurospora crassa, ectophosphatases, which are repressible by an excess of phosphate ions in the medium, apparently have the role of increasing the avail- ability of inorganic phosphate (Heredia et al. 1963; Nyc 1967). A repressible acid phosphatase has been isolated from the cell wall of Saccharo- inyces inellis (Weimberg and Orton 1964) and localized histochemically in yeast cell walls (Giinther et al. 1967). In the present study, stain- ing was observed in the spore walls and most probably the walls contain an acid phosphatase which may also function in increasing the avail- ability of inorganic phosphate.

With our incubation procedures stain was variablv associated with nuclei in both the test and control sections of the plasmodium. The spores lack dense nuclear stains but diffuse stain- ing of the nucleus was present in some controls. It may be that some of the plasmodial sections were over-incubated. In over-incubated material the primary and final-reaction product may dif- fuse through the material and concentrate at ., non-enzymic sites, especially the nucleus (Gold- fischer et al. 1964; Shnitka and Seligman 1971). The accuracy of acid phosphatase localization, using the lead-salt method, depends on the prox- imity of the enzyme to concentrations of lead- attracting material. The reaction product is adsorbed probably by a phospholipid or phos- pholipid complex (Hayashi 1971). The stain in the nuclei of the plasmodial controls may be due to nonspecific binding of lead. Since the fruiting bodies of P. vermicularis were incubated intact to prevent dissipation of the spores, the nuclear stain may be an artifact resulting from the thick- ness of the tissue. Uniform staining was obtained by Holt and Hicks (1961) using sections of 50 p or less of rat liver tissue. Holt and Hicks (1961) stated that the artifacts in thick sections result from different rates of ~enetration of the com- ponents in the medium. The solution to penetra- tion problems appears to be the use of very thin sections for localization experiments (Sexton et al. 1971). Nuclear staining may also result from the use of old staining medium, which contains less lead than fresh solutions (Holt 1959). Other possibilities which may produce nuclear staining, including glutaraldehyde activation of P-glycero- phosphatase (De Jong et al. 1967), movement of cytoplasmic enzymes during fixation, and endo-

genous substrate, have not been examined in this material. Not all staining of nuclei is artifactual however; high P-glycerophosphatase activity has been obtained in nuclei of cortical root cells in maize and pea (Sexton et al. 1971) and in Hela cells (Soriana and Love 1971). Additional experimentation is needed to determine if nuclei of P. vermicularis stain positive for P-gly- cerophosphatase.

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CRONSHAW A N D CHARVAT: PERICHAENA VERMICULARIS 101

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EXPLANATION OF FIGURES KEY TO ABBREVIATIONS: LD, lipid droplet; M, mitochondria; N, nucleus; PM, plasma membrane; SW,

spore wall; and V, vacuole. FIG. 1. Perichaena vermicrrlaris. p-Glycerophosphatase localization. Plasmodium incubated in medium

with substrate for 75 min. Reaction product is localized in the vesicles, at the edge and inside the nuclei, and in some of the mitochondria. Stained with uranyl acetate and lead. X 8500.

FIG. 2. Perichaet~a vermicrrla~~ir. Control: no substrate present. Incubated for 90 min. Cross section of the plasmodium. Only a small amount of non-enzymatic stain is present. Unstained. X 7000.

FIG. 3. Perichaetra vermicularis. Control: no substrate present. Incubated for 90 min. Cross section of the plasmodium from the same block as Fig. 2. Non-enzymatic localization of lead phosphate in the nucleus and the cytoplasm of the induced plasn~odiunl. Stained with uranyl acetate and lead. X 8500.

FIG. 4. Perichaena vermicrrlaris. Incubated in medium with substrate for 105 min. Cross section of dif- ferentiating fruiting body. 13-Glycerophosphatase is localized in the large and small vacuoles of the cleaved protoplasm. Some of the nlitochondria are also stained. Stained with uranyl acetate and lead. X 15 000.

FIG. 5. Perichaena vermicrilaris. Fruiting body incubated in medium with substrate for 105 min. Reaction product is localized in the single large vacuole of the mature spore. No nuclear staining is present. Stained with uranyl acetate and lead. X 14 200.

FIG. 6. Perichaetra vermicrilaris. Longitudinal section of a 2-week-old spore. Incubated in medium with substrate for 70 min. The reaction product is localized in the central vacuole and around the wall. Diffuse staining is present throughout the rest of the cytoplasm. Stained with uranyl acetate and lead. X 13 900.

NOTE: Figs. 1-6 follow.

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