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J. Cell Set. 51, 15-23 (1981) Printed in Great Britain © Company of Biologist! Limited ig8i 15 ACID PHOSPHATASE LOCALIZATION IN PAS-BODIES OF GONYAULAX RUTH E. SCHMITTER AND ANTONI J. JURKIEWICZ Biology Department, University of Massachusetts at Boston, Boston, Mass. 02125, U.S.A. SUMMARY Periodic acid-Schiff staining, acid phosphatase localization, and yellow autofluorescence have been correlated with the PAS-body of Gonyaulax polyedra for the first time. PASstaining and acid phosphatase activity are both correlated with the PAS-body of Gonyaulax tamarensis. These results suggest that the PAS-body of these marine dinoflagellate algae functions in subcellular digestion. INTRODUCTION It is important to know the subcellular localization of acid phosphatase activity in algal cells. This enzyme is associated with digestive processes throughout the animal kingdom, but there are fewer reports of subcellular localizations from plant cells, and especially from the algae. Many of the algal organisms studied were in unusual nutrient conditions or undergoing specific developmental changes. These include 2 species of Euglena under conditions of carbon starvation and aging (Brandes, Buetow, Bertini & Malkoff, 1964; Malkoff & Buetow, 1964; Brandes, 1965; Sommer & Blum, 1965; Palisano & Walne, 1972; Gomez, Harris & Walne, 19740,6); 3 species of Cryptomonas (Lucas, 1970); Polytomella caeca, a colourless heterotroph (Cooper, Bowen & Lloyd, 1974); Viva mutabilis during gamete release and fertilization (Briten, 1975) and U.lactuca vegetative cells (Micalef, 1975); Micrasterias americana at various stages of growth (Noguchi, 1976); aging Ectocarpus sp. (Oliveira & Bisalputra, 1977); and 2 wall-less volvocine algae, Dunaliellaprimolecta (Eyden, 1975) and Asteromonas gracilis (Swanson & Floyd, 1979). Cultured Gonyaulax polyedra cells possess a large membrane-bounded spherical body, portions of which are stained by the periodic acid-Schiff (PAS) reaction. Schmitter (1971) proposed an active digestive function for these PAS-bodies, either in autophagy or in the use of stored metabolites, based in part on their ultrastructural contents: aggregates of electron-dense material, fibrous areas, and membranous vesicles. PAS-bodies are morphologically similar to the digestive granules in absorptive cells of Hydra (Slautterback, 1967) and to the food vacuoles of the dinoflagellates Ceratium hirundinella and Amyloodinium sp. (Dodge & Crawford, 1970; Lorn & Lawler, 1973). They are also similar in many respects to the Corps de Maupas of Cryptomonas reticulata (Lucas, 1970). They bear a lesser structural resemblance to the 'accumulation bodies' of symbiotic dinoflagellates (Taylor, 1968, 1971a, b\ Tomas & 16 R. E. Schmitter and A. J. Jurkiewicz Cox, 1973; Trench, 1974), in which granular electron-dense material increases with cell age until the contents often appear quite homogeneous in texture. Taylor (1968) suggested that the structure is an accumulation site for waste materials and Tomas & Cox (1973) proposed that the accumulation bodies of Peridinium balticum function in waste storage or elimination. Lee (1977) has described accumulation bodies in freeliving colourless heterotrophic isolates of Gyrodinium lebouriae Herdman but these resemble PAS-bodies structurally more than accumulation bodies. In our study we found acid phosphatase activity to be localized in the PAS-bodies of the red tide organisms, Gonyaulax polyedra and G. tamarensis. This investigation also supports the proposal of Schmitter (1971, 1973) that PAS-bodies of G. polyedra have a digestive function. MATERIALS AND METHODS Growth of cells G. polyedra and G. tamarensis were cultured in medium f/2 without silicate (Guillard & Ryther, 1962) at 21 and 16 °C, respectively, under an alternating cycle of 12 h light-12 h dark. G. polyedra strain GP60E was obtained from Dr R. R. L. Guillard, Woods Hole Oceanographic Institution; G. tamarensis was isolated by Dr C. Martin, University of Massachusetts Marine Station, Gloucester. All samples used were from cultures in the first hour of their light period (CT 0-1) unless otherwise stated. Acid phosphatase localization Cells were collected by centrifugation at 80-100g in an IEC clinical centrifuge for 4 min, then fixed with 2-5% glutaraldehyde in 005 M-sodium cacodylate buffer (pH 7-2), for 30 min at 3-4 °C. Subsequent steps were carried out in 12-ml centrifuge tubes; centrifugation between steps was at 80-100g for 4 min. Acid phosphatase was localized within whole cells by a modification of Trelease's method (1980) for algae. Fixed cells were rinsed 3 times in cacodylate buffer at room temperature before preincubating in 005 M-sodium acetate buffer (pH 5-o) for 20 min at room temperature. The acid phosphatase medium was mixed as follows to minimize precipitation: 0-36 M-lead nitrate was prepared using cooled, freshly boiled distilled water. One ml of lead nitrate solution was added in 20-fi\ aliquots with gentle sirring to n o ml of 0-05 M-sodium acetate buffer (pH 5-0) in which 0-3 g of sodium /?-glycerophosphate had just been dissolved. The complete medium was preincubated at 37 °C for 1 h and any precipitate removed using Whatman no. 1filterpaper. Controls consisted of incubation medium lacking substrate (Pb1+ control) and complete incubation medium containing the enzyme inhibitor sodium fluoride (NaF control). In the latter, sodium fluoride was added to the buffersubstrate mixture to give a final concentration of 001 M before lead nitrate was added. Control solutions were also preincubated and filtered. All incubations were carried out as 5 -ml volumes in stoppered centrifuge tubes for 30 min at 37 °C with occasional gentle stirring. The ensuing steps were at room temperature. Cells were rinsed 3 times in distilled water, soaked for 5 min in i - o% acetic acid to remove unprecipitated lead, and rinsed again in distilled water. Sites of lead phosphate deposition were revealed by conversion to lead sulphide using i - o% aqueous ammonium sulphide for 10 min. Cells were rinsed thoroughly with distilled water before mounting in glycerol for light microscopy. Some preparations were stained with acetocarmine, so that nuclei were also clearly visible. Cells were examined by light microscopy for acid phosphatase localization at a magnification of x 400. Cells from several adjacent fields were scored for lead sulphide deposition within PAS-bodiea; a minimum of 500 cells was counted from each slide. Photographs were recorded on Kodak Plus-X-Pan or Ektachrome 200 film. Acid phosphatase in Gonyaulax 17 Periodic acid-Schiff staining Periodic acid-Schiff (PAS) staining was carried out as described by Grimstone & Skaer (1972); their directions for Schiff's reagent were also used. Cells were fixed as for the acid phosphatase studies, or at room temperature in ethanol/glacial acetic acid (3:1, v/v) for 30 min. Cells were rinsed thoroughly after either fixation. Non-specific staining of glutaraldehyde-fixed cells was prevented by blocking with aniline/glacial acetic acid (1 :<), v/v) for 20 min. Pellets were gently resuspended throughout using a Pasteur pipette. Oxidation, staining, and rinsing times were as described by Grimstone & Skaer, except that rinses were done by repeated centrifugation. Cells were mounted in glycerol and examined for PAS staining at a magnification of x 400. Fluorescence microscopy Autofluorescence of the PAS-bodies was studied using either unfixed cells, or cells fixed in the ethanol/acetic acid fixative just described. Fixed cells were rinsed in distilled water twice and mounted in glycerol for study with a Zeissfluorescencemicroscope equipped with a highintensity illuminator and superpressure mercury vapor lamp HBO 200 W/4. Excitation filters BG3 and UGi were used in conjunction with a no. 53 barrier filter. Photographic records were made using Kodak Technical Pan Film 2415 (ESTAR-AH base) and developed for 4 min with Kodak D-19 developer for maximum contrast. At least 500 cells were examined at a magnification of x 400 in all studies. The autofluorescence is yellow. RESULTS Acid phosphatase The highest levels of acid phosphatase activity were recorded when 2-5% glutaraldehyde was employed. Lower concentrations of glutaraldehyde resulted in relatively low levels of localization in identifiable PAS-bodies. For example, after 1*5% Table 1. Acid phosphatase localization in Gonyaulax % Acid phosphatase in PAS-bodies Organism G. polyedra G. tamarensis (days) Experimental Pb 1+ control NaF control 14 17 29 24 23 0 0 0 0 33 0 0 3° 24 0 0 35 45 0 0 21 13 0 1 glutaraldehyde fixation a maximum of 9% of cells examined showed acid phosphatase localization in PAS-bodies; Pb 2+ controls gave up to 1% positive results. In our hands methods other than that of Trelease (1980) resulted in a considerable degree of lead deposition within nuclei (e.g. see Gomori, 1952; Lewis & Knight, 1977). We did not use any substrates other than /?-glycerophosphate in our studies (see Beaufay, 1972). Table 1 lists the results of several acid phosphatase localization experiments using G. polyedra and one using G. tamarensis. The percentage of cells with lead sulphide deposits in identifiable PAS-bodies is given for cultures of several ages. Figs. 4 and 7 R. E. Schmitter and A. J. Jurkicuricz Acid phosphatase in Gonyaulax 19 show typical examples of localizations in G. polyedra; Fig. 3 gives a comparable picture of G. tamarensis. Figs. 1 and 2 depict typical cells from NaF and Pb 2+ controls in G. tamarensis and G. polyedra, respectively. The data do not allow any conclusions about the relative amounts of acid phosphatase activity present in PAS-bodies of cells from cultures of different ages. Some of the reasons for this are discussed below. A portion of cells in all experimental preparations stained non-specifically. We do not know whether this reflects intracellular disruption or some other undetermined variable. Such ' overstaining' was never observed in the NaF or Pb 2+ controls. Cells from the 29-day culture experiment were stained by the PAS reaction after aniline blockage. A total of 35% of the experimental cells contained PAS-reactive PAS-bodies, as did 35% of the Pb2+ control cells. The reasons for the lower percentage of localization in G. tamarensis are unknown. That experiment was done using the same incubation mixture as the 29-day G. polyedra culture. Fluorescence Fluorescent PAS-bodies were seen in a high percentage of G.polyedra cells examined from 3 times of day (14-day culture). Circadian times o and 7 h (light cycle), and 16 h (dark cycle) had 90, 99, and 89% yellow autofluorescent PAS-bodies, respectively. Cells from o h showed no fluorescence at all if subjected to the PAS reaction before viewing. A total of 98 % of these same cells had PAS-bodies when viewed by conventional illumination. Fig. 5 depicts cells of G. polyedra as viewed by conventional illumination; Fig. 6 shows the same cells as viewed by fluorescence microscopy. The PAS-bodies are clearly visible as discrete sites of fluorescence surrounded by indistinct cytoplasm. Fig. 1. G. tamarensis; NaF control. Only 1 % of NaF control cells showed any lead sulphide deposition. Arrow indicates girdle region of cell. Clear area within is the nucleus. Figs. 1-5, bar 20/tm. Fig. 2. G. polyedra; Pb l + control. None of the Pb l + control cells showed lead sulphide deposition in any of the experiments described. Fig. 3. G. tamarensis; acid phosphatase experimental preparation of 21-day culture. A total of 13 % of cells had lead sulphide localized in a PAS-body. Arrows indicate deposits. Fig. 4. G. polyedra; acid phosphatase preparation of 35-day culture. A total of 45 % of cells had lead sulphide localized in PAS-bodies. Arrows indicate 3 deposits. Fig. 5. G. polyedra viewed by conventional illumination after fixation with ethanol/ acetic acid. Fig. 6. Same cells as those of Fig. 5, viewed by fluorescence microscopy; 7-5 min photographic exposure time. Discrete sites of fluorescence (PAS-bodies) are clearly visible, as are outlines of surrounding cytoplasm. R. E. Schmitter and A. J. Jurkieioicz 2O er Fig. 7. Two cells of G. polyedra with acid phosphatase localization in PAS-bodies. PAS-body in cell at right shows typical compact morphology. That at upper left has several large granules at its periphery (arrows). Bar, 10 fim. Fig. 8. PAS-body of G. polyedra by electron microscopy. Methods were as given by Schmitter (1971). Two membrane-bound elements (1, 2) are closely associated with the PAS-body, and another (3) is fusing with it. These elements may correspond to the granular localizations noted in Fig. 7. ch, chloroplast; st, starch; er, endoplasmic reticulum. Bar, 1 fim. Acid phosphatase in Gonyaulax 21 DISCUSSION Results of the acid phosphatase studies show substantial localization within PASbodies. These results are not completely quantitative, however. First, it is known that glutaraldehyde inhibits a portion of acid phosphatase activity, while at the same time preserving sufficient activity for cytochemical localization (Sabatini, Bensch & Barrnett, 1963; Brunk & Ericsson, 1972). Swanson & Floyd (1979) determined from in vitro studies of the /?-glycerophosphatase from Asteromonas that about 70% of the activity of the enzyme was lost after the glutaraldehyde fixation method they employed for cytochemistry. We have not done such a study, but the amounts of activity localizable in G. polyedra are comparable to the amount of activity remaining in their study. Second, we have not determined the pH optimum for acid phosphatase activity in G. polyedra. If it differs very much from pH 5-0, localizable activity would be lower than the optimum. Although we have not seen a study in which the pH optimum for acid phosphatase activity was much higher, Miiller (1973) has described acid phosphatase activity from a trichomonad flagellate, with substantial activity above pH 6. Third, because we used whole cells in our study, we cannot be certain that small deposits of lead sulphide would be visible by light microscopy. If elements such as those associated with the PAS-body in Fig. 8 (labelled 1, 2, 3) contained activity and the body-proper did not, we would not have been able to resolve them. Similarly, the contents of PAS-bodies as seen by electron microscopy are not homogeneous. Acid phosphatase activity might be present only in certain portions at any given time. Small localizations would be visible only by electron microscopy. It is possible that the granular deposits associated with the PAS-body in Fig. 7 do represent large membranous elements similar to those seen at the periphery of the PAS-body in Fig. 8. The vegetative nucleus of G. polyedra is C-shaped, and the Golgi dictyosomes are arranged within the inner curve of the nucleus (Schmitter, 1971). Occasionally, we observed granular deposits between the PAS-body and the inner curve of the nucleus. We are currently doing electron microscopic studies to investigate this point. Correlations between autofluorescent cytoplasmic granules and lysosomal activity have been made in a number of animal tissues (Koenig, 1963; Strehler, 1977). Pearse (1968) tentatively attributed this autofluorescence either to lipid or lipoprotein of lysosomal membranes, or to lipid material dissolved inside the organelles. Correlations have also been made between lysosomal activity and PAS-reactive material (Koenig, 1962; Strehler, 1977), and Koenig reported the disappearance of autofluorescence of lysosomes in certain tissues after the PAS-reaction. Studies on lipofuscin pigments in aging mammalian tissues (summarized by Strehler, 1977) have linked lysosomal PASreactivity and autofluorescence with the presence of partially oxidized, non-carbohydrate-containing lipids (Pearse, 1972). The fragments of membrane seen within PAS-bodies in our study (Fig. 8; also by Schmitter, 1971) support the notion that lipid composition of PAS-bodies is related to their autofluorescence and PASreactivity. PAS-staining and acid phosphatase localization were observed by us in the same 22 R. E. Schmitter and A. J. Jurkievdcz PAS-bodies of G. polyedra when cells were processed sequentially for acid phosphatase localization and PAS-staining. Moreover, the size and unique location of PAS-bodies within G. polyedra make it possible to correlate PAS-staining, acid phosphatase activity, and autofluorescence unequivocally. Gomez et al. (1974 a) unsuccessfully attempted a similar correlation in aging Euglena gradlis. Their Euglena cells had been treated with benzpyrene, so they were observing secondary lipid fluorescence, not autofluorescence (Pearse, 1968). Our studies have shown that the PAS-bodies are a relatively permanent fixture in G. polyedra. Their acid phosphatase activity is probably, as originally proposed by Schmitter (1971), employed in autophagic processes recycling cellular materials. The only other alga so far reported to possess a permanent site for digestive processes, as opposed to the more usual transitory lysosomal activity, is Cryptomonas. Lucas (1970) localized acid phosphatase activity in the Corps de Maupas and has suggested that the structure is permanent, since it can be observed in natural populations and cultures under a variety of conditions. Lucas (1970) did not report any studies of PAS-staining or autofluorescence of this structure. Our study is the first report in which PAS-staining, acid phosphatase localization, and autofluorescence have all been correlated with the same subcellular algal organelle. REFERENCES BEAUFAY, H. (1972). The non-lysosomal localization of acid (p-nitro)-phenyl phosphatase activity in various tissues. Appendix I to Methods for the Isolation of Lysosomes. 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