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Estimation of vacuolar pH in actively growing hyphae of the fungus Pisolithus tinctorius
Myco/. Res. 99 (5): 549-553 (1995)
549
Printed in Great Britain
Estimation of vacuolar pH in actively growing hyphae of the fungus Pisolithus tinctorius
F. W. D. ROSTl, V. A. SHEPHERDz AND A. E. ASHFORD z Schools of lAnatomy and 2Biological Science, University of NSW, Kensington, NSW 2033, Australia
The pH of vacuoles in tip cells of growing hyphae of a mycorrhizal fungus, Pisolithus tinctorius, was estimated by dual-wavelength microspedrofluorometry of 6-carboxyfluorescein. The vacuoles were found to have a modal pH of 7'0-7'5 and a pH range of 4'3-7'5. Vacuoles in penultimate cells tended to be more acid, with a modal group pH 5'5--6'0, although the range was similar at pH 4'8-7'2. The findings are discussed in relation to endocytosis and ageing of vacuoles in P. tinctorius.
There have been many attempts at estimation of intracellular pH in fungi (see Borst-Pauwels, 1981; Klionsky, Herman & Emr, 1990). Many of these have provided only a mean pH value for whole cells and have not distinguished sub-cellular compartments of potentially different pH (see Guern et a/., 1991). Actively growing hyphal tips of the mycorrhizal fungus Pisolithus tinctorius (Pers.) Coker & Couch contain a dynamic and pleiomorphic system of vacuoles and tubules which are thought to be involved in transport of phosphorus, as polyphosphate (Orlovich & Ashford, 1993; Shepherd, Orlovich & Ashford, 1993a, b; Ashford, Ryde & Barrow, 1994). This vacuolar system accumulates 6-carboxyfluorescein (CF) supplied to the cells as the diacetate. The appearance and behaviour of the vacuolar system in the hyphal tips is similar to that of endosomes and tubular lysosomes of endocytic systems as Seen in cultured animal cells (Tooze & Hollinshead, 1991; Shepherd ef al., 1993 a, b). It is possible that components of the vacuole system are receptive compartments for endocytosis, though endocytosis in fungal cells has been difficult to prove (as with other walled cells, see Robinson & Hillmer, 1990), because molecular markers large enough to be visible in the electron microscope are excluded by the wall, and uptake of smaller fluorochromes is not definitive (Preston, Murphy & Jones, 1987; Cole et al., 1991). We have, therefore, attempted to characterize these fungal compartments and compare them with endosomal systems of cultured animal cells (Ashford & Orlovich, 1994). Hydrogen ion concentration plays an important role in molecular sorting in both endocytic and exocytic pathways, allowing incoming receptors, ligands and flUid-phase components to display different properties in intracellular and extracellular compartments (Mellman, Fuchs & Helenius, 1986). Average pH values for whole cells are of little value in understanding this process, since it is the pH of individual compartments that matters. We have utilized the pH dependence of fluorescence of CF taken up into vacuoles to determine the individual pH values of the larger vacuoles in the motile vacuolar system of P.
tinctorius hyphal tips. The technique of measuring intracellular pH by microspectrofluorometry of fluorescent probes is now well established (Tsien, 1989) and CF fluorescence has previously been used to determine average pH values for yeast cells using flow cytometry (Preston, Murphy & Jones, 1989). The fluorescence of fluorescein in solution varies with pH, due to change in predominant ionic species from the cation (at pH 0), to the neutral molecule (at pH 3'3), the monoanion (at pH 5'5) and finally the dianion (at pH 12) (Martin & Lindqvist, 1975). Both excitation and (to a lesser extent) emission spectra change with pH. This enables the pH to be estimated by measuring the ratio of fluorescence at two excitation wavelengths. CF has a strong green fluorescence, similar to that of fluorescein, and a pKa of 6-4-6'5 (Tsien, 1989). CF will not normally cross the plasmalemma (Goodwin, 1983) but can be loaded into cells as the sparingly fluorescent 6-carboxyfluorescein diacetate (CFDA), which is cleaved by intracellular esterases to form the highly fluorescent CF (Goodall & Johnson, 1982). The advantage of this method is that it enables measurement of pH at specific locations within the vacuole system.
MATERIALS AND METHODS Fungal cultures
Pisolifhus tinctorius (isolate Dr-IS, see Grenville, Peterson & Ashford, 1986) was cultured on modified Melin-Norkrans agar medium (Marx 1969) at 22° in the dark for 1-3 wk with the following variations: I % agar, 1% o-glucose instead of sucroSe, light dried malt extract instead of paste and 0'003 % ferric citrate instead of ferric chloride. The agar growth medium had an initial pH of 5'0. During the growth period the colony expanded at a slow, consistent rate of 3 mm d- l and was subcultured every 3-4 wk. Measurements of vacuolar pH Were made on hyphal tips during this actiVe growth period, at the stage when the colony occupied about one third of the culture plate and the hyphal tips were
Estimation of pH in fungal vacuoles expanding into new areas of the medium. A wedge-shaped piece of the mycelium was cut from the edge of the culture using a razor blade, and floated agar-side uppermost in the fluorochrome solution. The mycelial wedge was sufficiently large that the cells in the centre remained viable for the duration of the experiment (up to ~ 4 h); cells of smaller wedges were less viable. The viability of cells to be measured was judged in a number of ways: (i) presence of cytoplasmic streaming; (ii) general appearance of the cytoplasm; (iii) absence of plasmolysis and lack of change in the morphology of the hyphal tips; (iv) appearance and activity of the vacuole system itself. The hyphal tips examined were those growing just beneath the surface of the agar, in advance of the aerial hyphae.
Loading cells with 6-carboxyfluorescein Stock solutions of 6-carboxyfluorescein diacetate (CFDA), 'isomer free' (cat no. C1362), obtained from Molecular Probes, OR, U.S.A., were made up at 1 g 1-1 in acetone and stored in the dark. A 20 ~g ml- 1 solution was made up by diluting this with reverse osmosis water (final pH 4'8). Two to three hyphal wedges in agar were added to about 4 ml CFDA solution in a small Petri dish. The solution was not buffered because addition of either phosphate or zwitterionic buffers was found to diminish cell viability and a very dilute solution reflects the growth conditions used. Loss of viability was indicated by cessation of cytoplasmic streaming, swelling of the hyphal tips and characteristic changes in the appearance and motility of the vacuole system (see Shepherd et al., 1993a). Cells measured were all deemed viable based on the above criteria. The pH of the solution at the end of the experiment was essentially unchanged. The CFDA solution had very low fluorescence for at least 24 h, indicating a low CFDA hydrolysis rate. The optimal time-course for loading of the fluorochrome was a 10 min pulse with CFDA followed by a 30 min immersion in distilled water.
Test for identity of compartmental fluorochrome CF was obtained by hydrolysis, by allowing the unbuffered CFDA solution to stand for about 7 d at about 21° in the dark, by which time it had become very fluorescent. This solution and fresh CFDA solution were compared by thin layer chromatography with homogenates of untreated fungal hyphae and of hyphae loaded with CFDA as described above. Plates (Kieselgel 60; Merck. Darmstadt, Germany) were developed with n-butanol: acetic acid: pyridine: water, 15: 3: 10: 12 by volume, and examined for fluorescence under uv light. All solutions, except the untreated fungal homogenates, gave a single green-fluorescent spot, Rr 0'77, indicating that the reagents did not contain other fluorescent compounds that might interfere with the assay, and that the accumulated fluorochrome was CF. The freshly prepared CFDA showed only a faint spot at Rf 0'77, indicating that it contained a small amount of the hydrolysis product. The fungal homogenates, both control and CFDA-treated, additionally showed two pale blue fluorescent spots, with Rf values of 0'85 and 0'72, presumed to be due to autofluorescent tissue substances.
550
Microfluorometry The microfluorometer was based on a Leitz Orthoplan microscope, with an MPY-1 microphotometer (Leica Instruments and Systems, Wetzlar, Germany). A special stage was fitted, derived from a Barr & Stroud microdensitometer, with micrometer screw movements in x and y axes, to enable precise positioning of the specimen. Rotation of the photometer unit enabled a rectangular measuring slit to be orientated precisely over individual vacuoles. The measuring field typically corresponded to an area 5 x 10 ~m, and fields were chosen so that measurements of individual vacuoles could be made. Fluorescence excitation was obtained with a Ploemopak epi-illuminator and an HB0100 mercury arc lamp run from a stabilized DC power supply. Filtration of excitation and emission wavelengths was carried out by the standard filters in the Ploemopak epi-illuminator, namely Leica filter block I (blue excitation; barrier filter LP515; BP 450-490) and Leica filter block D (violet plus ultraviolet excitation; barrier filter LP460; BP 355-425). Photomultiplier output was measured with a digital voltmeter with 3'5 digits, reading to 10 mY across 10 megohms (equivalent to 1 nA). Measurements were made alternately with blue and violet excitation. To compensate for fading, sets of three measurements were made in the order blue, violet, blue; a geometric mean value for blue was calculated, on the assumption that fading would be approximately exponential (Rost, 1991). The ratio of fluorescence emission intensities with blue and violet excitation was calculated as a measure of pH. Experiments were carried out at 24° approx. and the duration of excitation was about 2-5 s per reading. Measurements were made of 1-4 vacuoles in each ceiL in either terminal or penultimate cells, totalling 103 sets of measurements in all. In addition, six successive measurements were made on a single vacuole over a 10 min period, the excitation being blocked off between measurements to reduce fading. Measurements were also made of consecutive vacuoles along the two or three cells of hyphal tips, in both directions. In the final analysis, values were rejected if (i) there was documented technical error, (ii) the second blue reading differed from the first by more than 10% (indicating excessive fading) or, (iii) any reading was less than 10 fluorescence units above the dark current.
Standardization Solutions of 6-carboxyfluorescein, 'isomer free' (cat no. C1360 lot no. 2522-3, Molecular Probes) at known pH were prepared in 50 mM potassium phosphate buffer or 50 mM potassium hydrogen phthalate buffer. These were calibrated using the HB0100 lamp with the microscope objective dipped into the solution in a well slide. A cubic regression curve was fitted to the data (Fig. 1). The regression curve was incorporated into a data reduction and statistical analysis program written in BASIC. The CF standard curve was in good agreement with a similar pH curve obtained from solutions prepared by hydrolysis of CFDA solutions from the same batch as that used in the vacuole measurements. The solution of CF in a buffer was regarded as a good model for intravacuolar CF, and it was not considered necessary to correct for in vivo measurements (d. Bright et aI., 1989;
551
F. W. D. Rost, V. A. Shepherd and A. E. Ashford
Ratio of fluorescence b/v Fig. 1. pH calibration curve for 6-carboxyfluorescein (cat no. C-1360 lot no. 2522-3, Molecular Probes). The ratio of fluorescence with blue excitation to that with violet excitation ('b/v') is shown.
Davies, Brownlee & Jennings, 1990). Dual labelling indicated that the fluorochrome was located in the vacuoles (Ashford & Orlovich, 1994) and, although hydrolases are likely to be present in some of the vacuoles, the levels of proteins and other macromolecules except polyphosphate were not sufficient to be detedable by histochemical staining (Orlovich & Ashford, 1993; Orlovich, unpublished observations; Ryde. unpublished observations). Moreover, Preston et al. (1989) found no evidence for perturbations of CF fluorescence by vacuolar constituents.
RESULTS The fluorochrome was rapidly sequestered into the vacuole system and. in those hyphae where the tip cell vacuole system was active and showed no indication of damage. there were
low to negligible levels of fluorescence in the cytoplasm. The only detectable autofluorescence was a faint orange in the walls of older cells under blue excitation. The hypha! tip cells did not show this and measurements on cytoplasmic areas not containing vacuoles gave readings not above the dark current. The vacuolar system showed variation in form, ranging from a tubular reticulum to a system predominantly of larger interconneded vacuoles, as described in Shepherd et ai. (1993a). Figure 2 shows vacuoles that are typical of those measured. Limitations of photometer sensitivity precluded measuring any but the larger vacuoles, and these tended to be at the basal end of terminal cells and throughout penultimate cells. Autofluorescence was not detected in the vacuoles at the excitation wavelengths used. The pH values obtained for the vacuoles measured are shown in Fig. 3. Acceptable pH estimates were obtained for 103 vacuoles, of which 68 were in 35 terminal cells and 35 vacuoles in 11 penultimate cells. Vacuoles in terminal cells (Fig. 3a) were found to have a pH in the range 4'3-7'5. with a modal group at pH 6'0-6'5. Vacuoles in penultimate cells (Fig. 3 b) were found to have a pH in the range 3'3-6'4, with no obvious modal group. The combined data are shown in Fig, 3 c and had a distribution biased towards the alkaline range, with modal group at pH 6'0-6'5 and a range pH 3'3-7'5. Values for vacuoles in some cells, when measured successively, showed a trend of reduction in vacuolar pH regardless of the diredion of measurement along the hypha, and successive measurements on one individual vacuole also showed progressive decrease in pH with time. This was not invariably the case, but in consequence it was thought best to analyse separately the data from the first vacuole measurement made in each cell (Fig. 3), a total of 43 vacuoles. The combined data for first-measured vacuoles in both cell types were again consistent with the sum of two populations, one (predominant in terminal cells) haVing a range pH 4'3-7'5 and a modal group pH 7'0-7'5, and the other (predominant in penultimate
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-
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Fig. 2. The vacuolar system of Pisolithus tinctorius hyphal tips. shOWing accumulation of 6-carboxyfluorescein. A. Terminal cell with a typical vacuole and tubule system. Arrows at (a) indicate the position of the hyphal tip, (b) marks branch points of the reticulum. (c) is a point where tips of two tubules have approached one another. B. The larger vacuoles (v) are typical of those measured, Note that many of these vacuoles have tubular extensions or are interconnected by tubules. Bar, 20 11m.
Estimation of pH in fungal vacuoles 30 "'T"""-----------..., A
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Fig, 3. Histogram of the distribution of pH in individual vacuoles.
Terminal cells, (B) penultimate cells, (C) combined data for both cell types. Data for the first-measured vacuole in each cell are shown with stippled bars. (A)
cells) with a similar range pH 4'8-7'2 and a modal group at pH 5'5-6'0. The modal group for terminal cells, as expected, was more alkaline than that of the total data set.
DISCUSSION Measurements of pH, to be biologically meaningfuL must be made in clearly defined regions of the cell. Averaging over two or more compartments, to say nothing of two or more cells, may indicate the general order of the [H+] but fails to reveal the spread of pH values across different compartments.
552
Moreover, it may not be possible even to compare' average' figures from different techniques, because the averaging may be based on different parameters. It must be remembered that pH is an arbitrary mathematical function of [H+], and that mean pH and mean [H+] from the same set of data do not correspond (see Table 1). For this reason we have presented the modal groups and ranges of the pH data in this paper rather than the means and have not generally compared our data with means obtained by others using different techniques. Comparison of the data in Fig. 3 suggests that there was indeed a tendency for the pH values to become less alkaline with sequential measurements. Whether this was due to a change in pH or to a deterioration of the fluorochrome with exposure is not known. It seemed appropriate, therefore, to accept only the first measurements on each vacuole as the best indication of the pH, and the discussion below is based on this premise. The wide range of pH values obtained in P. tinctorius supports the view that the vacuolar compartment does not show a uniform pH, but shows localized differences in [H+] concentration. It is unlikely there was a contribution of either the cytoplasm or wall to the reading, since readings on areas not containing vacuoles were not above the dark current. Addition of the CFDA solution and water rinse would be expected to dilute the Melin Norkrans medium (an already dilute nutrient solution). Since the conditions in nature for this fungus (i.e. Australian soils) are both low in nutrients and osmolarity these conditions should cause minimum perturbation of the hyphal tips. This was borne out by the hyphal appearance and maintenance of viability during the experiment. Since the first measurements of vacuoles in terminal cells had a modal group pH 7'0-7-5 and penultimate cells tended to be more acid (modal group pH 5'5-6'0) some of the variability could be explained by a progressive acidification with age of the cell. It is likely that acidification of the vacuole is brought about by membrane-bound H+-translocating ATPases that generate proton gradients across the tonoplast as found in other fungi (Bowman & Bowman, 1986; Mellman et aI., 1986; Rothman et al., 1989; Yamashiro et al., 1990). The modal pH groups in P. tinctorius are close to the values obtained by Makarow & Nevalainen (1987) in Saccharomyces cerevisiae for vacuoles under different conditions, using FITCdextran; their two sets of vacuoles had a mean pH of 5'8 (range 5-6-6'1, n = 8) and of pH 6-5 (range 6'2-6-7, n = 8) respectively. It is tempting to speculate that the two populations of vacuoles may represent two different compartments in the endocytic pathway, perhaps endosomes and Iysosomes, although the pH of the more alkaline set is above that normally expected for dissociation of receptor-ligand com-
Table 1. Comparison of mean pH and mean [H+] in mol 1-1 Parameter
Mean
Actual values (hypothetical)
[H']
I-a x 10-'
1'0 X 10- 6
pH
5'0
6'0
1'0
X
10- 7
I-a x 10-' 8-0
The mean [H+) corresponds to a pH of 5-56; the mean pH corresponds to [H+] = 3-2 x 10- 7 mol 1- 1 _
F. W. D. Rost. V. A Shepherd and A E. Ashford plexes in an endosomal compartment (Mellman ef a/., 1986), and it might be expected that the tubules and smaller vacuoles are the endosomes, rather than the vacuoles measured. Both sets of pH values are higher than those obtained for mammalian cells where Iysosomes are reported to be pH 4'6-5'0 and endosomes ~ pH 5'5 (see Mellman ef al., 1986). Endocytosis has not been proven definitively in P. fincforius,nor in any other fungus, but the vacuole and tubule system exhibits behaviour very much like endosomes or tubular Iysosomes as seen in mammalian tissue culture cells (Shepherd ef al., 1993 a; Ashford & Orlovich, 1994). It is noteworthy that the tubule and vacuole system is most active in the terminal cells, which would be the most likely of all the cells to be actively involved in endocytosis, and that the modal pH of vacuoles in these cells is in the more alkaline range. In contrast, the system is less mobile and larger vacuoles tend to predominate in the penultimate cells, which show the more acid vacuole population. Makarow & Nevalainen (1987) also suggested that their two types of vacuole may represent two compartments in the endocytotic system, but they suggested that the more acid compartments were the endosomes. Some doubt has been cast on their data, because of the impurities likely to be present in the FITCdextran (Preston ef al., 1987). Similar pleiomorphic vacuolar networks to that of P. fincforiushave been demonstrated in actively growing hyphal tips of a wide range of other fungi (Rees, Shepherd & Ashford, 1994) and it is clear that, at least in filamentous fungi, we can no longer consider fungal vacuoles as individuaL isolated organelles with a similar composition and pH. Rather, we should consider them as a composite series of interconnected compartments that may exhibit a wide range of pH values, possibly variable, but which are not acidified to the same extent as animal Iysosomes. The research was supported by grants from the National Health and Medical Research Council (equipment) to F. W. D. R. and from the Australian Research Council to AE.A We thank W. G. Allaway for comments and criticisms of the manuscript.
REFERENCES Ashford, A E. & Orlovich, D. A (1994). Vacuole transport, phosphorus, and endosomes in the growing tips of fungal hyphae. In Pollen-Pistil Interactions. Current Topics in Plant Physiology (ed. A G. Stephenson, T-h Kao). American Society of Plant Physiologists, Rockville, U.S.A (in the press). Ashford, A E., Ryde, S. & Barrow, K. D. (1994). Demonstration of a short chain polyphosphate in Pisolithus tinctorius and the implications for phosphorus transport. New Phytologist 126, 239-247. Borst-Pauwels, G. W. F. H. (1981). Jon transport in yeast. Biochimica et Biophysica Acta 650, 8&-127. Bowman, B. J. & Bowman, E. J. (1986). H+-ATPases from mitochondria, plasma membranes, and vacuoles of fungal cells. Journal of Membrane Biology 94,83-97. Bright, G. R., Fisher. G. W., Rogowska, J. & Taylor, D. L. (1989). Fluorescence ratio imaging microscopy. In Methods in Cell Biology, Vol. 30. Fluorescence Microscopy of Living Cells in Culture. Part B. Quantitative Fluorescence
(Accepted 30 September 1994)
553 Microscopy - Imaging and Spedroscopy (ed. D. L. Taylor & Y. L. Wang), pp. 157-192. Academic Press, Inc., San Diego, CA. Cole, L., Coleman,)., Keams, A., Morgan, G. & Hawes. C. (1991). The organic anion transport inhibitor, probenecid, inhibits the transport of lucifer yellow at the plasma membrane and the tonoplast in suspension-cultured plant cells. Journal of Cell Science 99, 545-555. Davies, J. M., Brownlee, C. & Jennings, D. H. (1990). Measurement of intracellular pH in fungal hyphae using BCECF and digital imaging microscopy. Evidence for a primary proton pump in the plasmalemma of a marine fungus. Journal of Cell Science 96, 731-736. Goodall, H. & Johnson, M. H. (1982). Use of carboxyfluorescein diacetate to study formation of permeable channels between mouse blastomeres. Nature 295, 524-526. Goodwin, P. B. (1983). Molecular size limit for movement in the symplast of the Elodea leaf. Planta 157, 124-130. Grenville, D. J., Peterson, R. L. & Ashford, A E. (1986). Synthesis in growth pouches of mycorrhizae between Eucalyptus pilulans and several strains of Pisolithus linctorius. Australian Journal of Botany 34, 95-102. Guem, )., Felle, H., Mathieu, Y. & Kurkdjian, A (1991). Regulation of intracellular pH in plant cells. International Review of Cytology 12 7, 111-173. Klionsky, D.)., Herman, P. K. & Emr. S. D. (1990). The fungal vacuole: composition, function and biogenesis. Microbiological Reviews 54, 266-292. Makarow, M. & Nevalainen, L. T. (1987). Transport of a fluorescent macromolecule via endosomes to the vacuole in Saccharomyces cerevisiae. Journal of Cell Biology 104, 67-75. Martin, M. M. & Lindqvist, L. (1975). The pH dependence of fluorescein fluorescence. Journal of Luminescence 10, 381-390. Mellman, I., Fuchs, R. & Helenius, A (1986). Acidification of the endocytic and exocytic pathways. Annual Review of Biochemistry 55, 663-700. Orlovich, D. A & Ashford, A E. (1993). Polyphosphate granules are an artefact of specimen preparation in the ectomycorrhizal fungus Pisolilhus tinctorius. Protoplasma 173,91-102. Preston, R. A, Murphy, R. F. & Jones, E. W. (1987). Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran. Journal of Cell Biology 105. 1981-1987. Preston, R. A, Murphy, R. F. & Jones, E. W. (1989). Assay of vacuolar pH in yeast and identification of acidification-defective mutants. Proceedings of the National Academy of Sciences U.SA. 86, 7027-7031. Rees, B., Shepherd, V. A & Ashford, A. E. (1994). Presence of a motile tubular vacuole system in different phyla of fungi. Mycological Research 98, 985-992. Robinson, D. G. & Hillmer, S. (1990). Endocytosis in plants. Physiologia Plantarum 79, 96-104. Rost, F. W. D. (1991). Quantitative fluorescence microscopy. Cambridge University Press: Cambridge, U.K. Rothman, J. H., Yamashiro, C. T, Raymond, C. T, Kane, P. M. & Stevens, T H. (1989). Acidification of the lysosome-like vacuole and vacuolar H+ ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins. Joumal of Cell Biology 109, 93-100. Shepherd, V. A, Orlovich, D. A & Ashford, A E. (1993 a). A dynamic continuum of pleiomorphic tubules and vacuoles in growing hyphae of a fungus. Journal of Cell Science 104, 495-507. Shepherd, V. A, Orlovich, D. A. & Ashford, A. E. (1993 b). Cell-to-cell transport via motile tubules in growing hyphae of a fungus. Journal of Cell Science 105. 1173-1178. Tooze, ). & Hollinshead, M. (1991). Tubular early endosomal networks in AtT20 and other cells. Journal of Cell Biology 115, 635-653. Tsien, R. Y. (1989). Fluorescent indicators of ion concentrations. In Methods in Cell Biology, Vol. 30. Fluorescence Microscopy of Living Cells in Culture. Part B. Quantitative Fluorescence Microscopy - Imaging and Spectroscopy (ed. D. L. Taylor & Y. L. Wang), pp. 127-156. Yamashiro, C. T, Kane, P. M., Wolczyk, D. F., Preston, R. A & Stevens, T H. (1990). Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar translocating ATPase. Molecular and Cellular Biology 10, 3737-3749.
549
Printed in Great Britain
Estimation of vacuolar pH in actively growing hyphae of the fungus Pisolithus tinctorius
F. W. D. ROSTl, V. A. SHEPHERDz AND A. E. ASHFORD z Schools of lAnatomy and 2Biological Science, University of NSW, Kensington, NSW 2033, Australia
The pH of vacuoles in tip cells of growing hyphae of a mycorrhizal fungus, Pisolithus tinctorius, was estimated by dual-wavelength microspedrofluorometry of 6-carboxyfluorescein. The vacuoles were found to have a modal pH of 7'0-7'5 and a pH range of 4'3-7'5. Vacuoles in penultimate cells tended to be more acid, with a modal group pH 5'5--6'0, although the range was similar at pH 4'8-7'2. The findings are discussed in relation to endocytosis and ageing of vacuoles in P. tinctorius.
There have been many attempts at estimation of intracellular pH in fungi (see Borst-Pauwels, 1981; Klionsky, Herman & Emr, 1990). Many of these have provided only a mean pH value for whole cells and have not distinguished sub-cellular compartments of potentially different pH (see Guern et a/., 1991). Actively growing hyphal tips of the mycorrhizal fungus Pisolithus tinctorius (Pers.) Coker & Couch contain a dynamic and pleiomorphic system of vacuoles and tubules which are thought to be involved in transport of phosphorus, as polyphosphate (Orlovich & Ashford, 1993; Shepherd, Orlovich & Ashford, 1993a, b; Ashford, Ryde & Barrow, 1994). This vacuolar system accumulates 6-carboxyfluorescein (CF) supplied to the cells as the diacetate. The appearance and behaviour of the vacuolar system in the hyphal tips is similar to that of endosomes and tubular lysosomes of endocytic systems as Seen in cultured animal cells (Tooze & Hollinshead, 1991; Shepherd ef al., 1993 a, b). It is possible that components of the vacuole system are receptive compartments for endocytosis, though endocytosis in fungal cells has been difficult to prove (as with other walled cells, see Robinson & Hillmer, 1990), because molecular markers large enough to be visible in the electron microscope are excluded by the wall, and uptake of smaller fluorochromes is not definitive (Preston, Murphy & Jones, 1987; Cole et al., 1991). We have, therefore, attempted to characterize these fungal compartments and compare them with endosomal systems of cultured animal cells (Ashford & Orlovich, 1994). Hydrogen ion concentration plays an important role in molecular sorting in both endocytic and exocytic pathways, allowing incoming receptors, ligands and flUid-phase components to display different properties in intracellular and extracellular compartments (Mellman, Fuchs & Helenius, 1986). Average pH values for whole cells are of little value in understanding this process, since it is the pH of individual compartments that matters. We have utilized the pH dependence of fluorescence of CF taken up into vacuoles to determine the individual pH values of the larger vacuoles in the motile vacuolar system of P.
tinctorius hyphal tips. The technique of measuring intracellular pH by microspectrofluorometry of fluorescent probes is now well established (Tsien, 1989) and CF fluorescence has previously been used to determine average pH values for yeast cells using flow cytometry (Preston, Murphy & Jones, 1989). The fluorescence of fluorescein in solution varies with pH, due to change in predominant ionic species from the cation (at pH 0), to the neutral molecule (at pH 3'3), the monoanion (at pH 5'5) and finally the dianion (at pH 12) (Martin & Lindqvist, 1975). Both excitation and (to a lesser extent) emission spectra change with pH. This enables the pH to be estimated by measuring the ratio of fluorescence at two excitation wavelengths. CF has a strong green fluorescence, similar to that of fluorescein, and a pKa of 6-4-6'5 (Tsien, 1989). CF will not normally cross the plasmalemma (Goodwin, 1983) but can be loaded into cells as the sparingly fluorescent 6-carboxyfluorescein diacetate (CFDA), which is cleaved by intracellular esterases to form the highly fluorescent CF (Goodall & Johnson, 1982). The advantage of this method is that it enables measurement of pH at specific locations within the vacuole system.
MATERIALS AND METHODS Fungal cultures
Pisolifhus tinctorius (isolate Dr-IS, see Grenville, Peterson & Ashford, 1986) was cultured on modified Melin-Norkrans agar medium (Marx 1969) at 22° in the dark for 1-3 wk with the following variations: I % agar, 1% o-glucose instead of sucroSe, light dried malt extract instead of paste and 0'003 % ferric citrate instead of ferric chloride. The agar growth medium had an initial pH of 5'0. During the growth period the colony expanded at a slow, consistent rate of 3 mm d- l and was subcultured every 3-4 wk. Measurements of vacuolar pH Were made on hyphal tips during this actiVe growth period, at the stage when the colony occupied about one third of the culture plate and the hyphal tips were
Estimation of pH in fungal vacuoles expanding into new areas of the medium. A wedge-shaped piece of the mycelium was cut from the edge of the culture using a razor blade, and floated agar-side uppermost in the fluorochrome solution. The mycelial wedge was sufficiently large that the cells in the centre remained viable for the duration of the experiment (up to ~ 4 h); cells of smaller wedges were less viable. The viability of cells to be measured was judged in a number of ways: (i) presence of cytoplasmic streaming; (ii) general appearance of the cytoplasm; (iii) absence of plasmolysis and lack of change in the morphology of the hyphal tips; (iv) appearance and activity of the vacuole system itself. The hyphal tips examined were those growing just beneath the surface of the agar, in advance of the aerial hyphae.
Loading cells with 6-carboxyfluorescein Stock solutions of 6-carboxyfluorescein diacetate (CFDA), 'isomer free' (cat no. C1362), obtained from Molecular Probes, OR, U.S.A., were made up at 1 g 1-1 in acetone and stored in the dark. A 20 ~g ml- 1 solution was made up by diluting this with reverse osmosis water (final pH 4'8). Two to three hyphal wedges in agar were added to about 4 ml CFDA solution in a small Petri dish. The solution was not buffered because addition of either phosphate or zwitterionic buffers was found to diminish cell viability and a very dilute solution reflects the growth conditions used. Loss of viability was indicated by cessation of cytoplasmic streaming, swelling of the hyphal tips and characteristic changes in the appearance and motility of the vacuole system (see Shepherd et al., 1993a). Cells measured were all deemed viable based on the above criteria. The pH of the solution at the end of the experiment was essentially unchanged. The CFDA solution had very low fluorescence for at least 24 h, indicating a low CFDA hydrolysis rate. The optimal time-course for loading of the fluorochrome was a 10 min pulse with CFDA followed by a 30 min immersion in distilled water.
Test for identity of compartmental fluorochrome CF was obtained by hydrolysis, by allowing the unbuffered CFDA solution to stand for about 7 d at about 21° in the dark, by which time it had become very fluorescent. This solution and fresh CFDA solution were compared by thin layer chromatography with homogenates of untreated fungal hyphae and of hyphae loaded with CFDA as described above. Plates (Kieselgel 60; Merck. Darmstadt, Germany) were developed with n-butanol: acetic acid: pyridine: water, 15: 3: 10: 12 by volume, and examined for fluorescence under uv light. All solutions, except the untreated fungal homogenates, gave a single green-fluorescent spot, Rr 0'77, indicating that the reagents did not contain other fluorescent compounds that might interfere with the assay, and that the accumulated fluorochrome was CF. The freshly prepared CFDA showed only a faint spot at Rf 0'77, indicating that it contained a small amount of the hydrolysis product. The fungal homogenates, both control and CFDA-treated, additionally showed two pale blue fluorescent spots, with Rf values of 0'85 and 0'72, presumed to be due to autofluorescent tissue substances.
550
Microfluorometry The microfluorometer was based on a Leitz Orthoplan microscope, with an MPY-1 microphotometer (Leica Instruments and Systems, Wetzlar, Germany). A special stage was fitted, derived from a Barr & Stroud microdensitometer, with micrometer screw movements in x and y axes, to enable precise positioning of the specimen. Rotation of the photometer unit enabled a rectangular measuring slit to be orientated precisely over individual vacuoles. The measuring field typically corresponded to an area 5 x 10 ~m, and fields were chosen so that measurements of individual vacuoles could be made. Fluorescence excitation was obtained with a Ploemopak epi-illuminator and an HB0100 mercury arc lamp run from a stabilized DC power supply. Filtration of excitation and emission wavelengths was carried out by the standard filters in the Ploemopak epi-illuminator, namely Leica filter block I (blue excitation; barrier filter LP515; BP 450-490) and Leica filter block D (violet plus ultraviolet excitation; barrier filter LP460; BP 355-425). Photomultiplier output was measured with a digital voltmeter with 3'5 digits, reading to 10 mY across 10 megohms (equivalent to 1 nA). Measurements were made alternately with blue and violet excitation. To compensate for fading, sets of three measurements were made in the order blue, violet, blue; a geometric mean value for blue was calculated, on the assumption that fading would be approximately exponential (Rost, 1991). The ratio of fluorescence emission intensities with blue and violet excitation was calculated as a measure of pH. Experiments were carried out at 24° approx. and the duration of excitation was about 2-5 s per reading. Measurements were made of 1-4 vacuoles in each ceiL in either terminal or penultimate cells, totalling 103 sets of measurements in all. In addition, six successive measurements were made on a single vacuole over a 10 min period, the excitation being blocked off between measurements to reduce fading. Measurements were also made of consecutive vacuoles along the two or three cells of hyphal tips, in both directions. In the final analysis, values were rejected if (i) there was documented technical error, (ii) the second blue reading differed from the first by more than 10% (indicating excessive fading) or, (iii) any reading was less than 10 fluorescence units above the dark current.
Standardization Solutions of 6-carboxyfluorescein, 'isomer free' (cat no. C1360 lot no. 2522-3, Molecular Probes) at known pH were prepared in 50 mM potassium phosphate buffer or 50 mM potassium hydrogen phthalate buffer. These were calibrated using the HB0100 lamp with the microscope objective dipped into the solution in a well slide. A cubic regression curve was fitted to the data (Fig. 1). The regression curve was incorporated into a data reduction and statistical analysis program written in BASIC. The CF standard curve was in good agreement with a similar pH curve obtained from solutions prepared by hydrolysis of CFDA solutions from the same batch as that used in the vacuole measurements. The solution of CF in a buffer was regarded as a good model for intravacuolar CF, and it was not considered necessary to correct for in vivo measurements (d. Bright et aI., 1989;
551
F. W. D. Rost, V. A. Shepherd and A. E. Ashford
Ratio of fluorescence b/v Fig. 1. pH calibration curve for 6-carboxyfluorescein (cat no. C-1360 lot no. 2522-3, Molecular Probes). The ratio of fluorescence with blue excitation to that with violet excitation ('b/v') is shown.
Davies, Brownlee & Jennings, 1990). Dual labelling indicated that the fluorochrome was located in the vacuoles (Ashford & Orlovich, 1994) and, although hydrolases are likely to be present in some of the vacuoles, the levels of proteins and other macromolecules except polyphosphate were not sufficient to be detedable by histochemical staining (Orlovich & Ashford, 1993; Orlovich, unpublished observations; Ryde. unpublished observations). Moreover, Preston et al. (1989) found no evidence for perturbations of CF fluorescence by vacuolar constituents.
RESULTS The fluorochrome was rapidly sequestered into the vacuole system and. in those hyphae where the tip cell vacuole system was active and showed no indication of damage. there were
low to negligible levels of fluorescence in the cytoplasm. The only detectable autofluorescence was a faint orange in the walls of older cells under blue excitation. The hypha! tip cells did not show this and measurements on cytoplasmic areas not containing vacuoles gave readings not above the dark current. The vacuolar system showed variation in form, ranging from a tubular reticulum to a system predominantly of larger interconneded vacuoles, as described in Shepherd et ai. (1993a). Figure 2 shows vacuoles that are typical of those measured. Limitations of photometer sensitivity precluded measuring any but the larger vacuoles, and these tended to be at the basal end of terminal cells and throughout penultimate cells. Autofluorescence was not detected in the vacuoles at the excitation wavelengths used. The pH values obtained for the vacuoles measured are shown in Fig. 3. Acceptable pH estimates were obtained for 103 vacuoles, of which 68 were in 35 terminal cells and 35 vacuoles in 11 penultimate cells. Vacuoles in terminal cells (Fig. 3a) were found to have a pH in the range 4'3-7'5. with a modal group at pH 6'0-6'5. Vacuoles in penultimate cells (Fig. 3 b) were found to have a pH in the range 3'3-6'4, with no obvious modal group. The combined data are shown in Fig, 3 c and had a distribution biased towards the alkaline range, with modal group at pH 6'0-6'5 and a range pH 3'3-7'5. Values for vacuoles in some cells, when measured successively, showed a trend of reduction in vacuolar pH regardless of the diredion of measurement along the hypha, and successive measurements on one individual vacuole also showed progressive decrease in pH with time. This was not invariably the case, but in consequence it was thought best to analyse separately the data from the first vacuole measurement made in each cell (Fig. 3), a total of 43 vacuoles. The combined data for first-measured vacuoles in both cell types were again consistent with the sum of two populations, one (predominant in terminal cells) haVing a range pH 4'3-7'5 and a modal group pH 7'0-7'5, and the other (predominant in penultimate
_ "-_-.-eB
~.o
-
~
Fig. 2. The vacuolar system of Pisolithus tinctorius hyphal tips. shOWing accumulation of 6-carboxyfluorescein. A. Terminal cell with a typical vacuole and tubule system. Arrows at (a) indicate the position of the hyphal tip, (b) marks branch points of the reticulum. (c) is a point where tips of two tubules have approached one another. B. The larger vacuoles (v) are typical of those measured, Note that many of these vacuoles have tubular extensions or are interconnected by tubules. Bar, 20 11m.
Estimation of pH in fungal vacuoles 30 "'T"""-----------..., A
25 20 15
10 5
o ...L...,.....-~a:.;,:.&...to:t_I~oL.I.:o;.:u::.;.:LJt.:,:oL.1o:fo'~.u 30 "'T"""-----------..., B
25
.... ~ 8 10 ;:l
Z 5
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c
25 20 15
Fig, 3. Histogram of the distribution of pH in individual vacuoles.
Terminal cells, (B) penultimate cells, (C) combined data for both cell types. Data for the first-measured vacuole in each cell are shown with stippled bars. (A)
cells) with a similar range pH 4'8-7'2 and a modal group at pH 5'5-6'0. The modal group for terminal cells, as expected, was more alkaline than that of the total data set.
DISCUSSION Measurements of pH, to be biologically meaningfuL must be made in clearly defined regions of the cell. Averaging over two or more compartments, to say nothing of two or more cells, may indicate the general order of the [H+] but fails to reveal the spread of pH values across different compartments.
552
Moreover, it may not be possible even to compare' average' figures from different techniques, because the averaging may be based on different parameters. It must be remembered that pH is an arbitrary mathematical function of [H+], and that mean pH and mean [H+] from the same set of data do not correspond (see Table 1). For this reason we have presented the modal groups and ranges of the pH data in this paper rather than the means and have not generally compared our data with means obtained by others using different techniques. Comparison of the data in Fig. 3 suggests that there was indeed a tendency for the pH values to become less alkaline with sequential measurements. Whether this was due to a change in pH or to a deterioration of the fluorochrome with exposure is not known. It seemed appropriate, therefore, to accept only the first measurements on each vacuole as the best indication of the pH, and the discussion below is based on this premise. The wide range of pH values obtained in P. tinctorius supports the view that the vacuolar compartment does not show a uniform pH, but shows localized differences in [H+] concentration. It is unlikely there was a contribution of either the cytoplasm or wall to the reading, since readings on areas not containing vacuoles were not above the dark current. Addition of the CFDA solution and water rinse would be expected to dilute the Melin Norkrans medium (an already dilute nutrient solution). Since the conditions in nature for this fungus (i.e. Australian soils) are both low in nutrients and osmolarity these conditions should cause minimum perturbation of the hyphal tips. This was borne out by the hyphal appearance and maintenance of viability during the experiment. Since the first measurements of vacuoles in terminal cells had a modal group pH 7'0-7-5 and penultimate cells tended to be more acid (modal group pH 5'5-6'0) some of the variability could be explained by a progressive acidification with age of the cell. It is likely that acidification of the vacuole is brought about by membrane-bound H+-translocating ATPases that generate proton gradients across the tonoplast as found in other fungi (Bowman & Bowman, 1986; Mellman et aI., 1986; Rothman et al., 1989; Yamashiro et al., 1990). The modal pH groups in P. tinctorius are close to the values obtained by Makarow & Nevalainen (1987) in Saccharomyces cerevisiae for vacuoles under different conditions, using FITCdextran; their two sets of vacuoles had a mean pH of 5'8 (range 5-6-6'1, n = 8) and of pH 6-5 (range 6'2-6-7, n = 8) respectively. It is tempting to speculate that the two populations of vacuoles may represent two different compartments in the endocytic pathway, perhaps endosomes and Iysosomes, although the pH of the more alkaline set is above that normally expected for dissociation of receptor-ligand com-
Table 1. Comparison of mean pH and mean [H+] in mol 1-1 Parameter
Mean
Actual values (hypothetical)
[H']
I-a x 10-'
1'0 X 10- 6
pH
5'0
6'0
1'0
X
10- 7
I-a x 10-' 8-0
The mean [H+) corresponds to a pH of 5-56; the mean pH corresponds to [H+] = 3-2 x 10- 7 mol 1- 1 _
F. W. D. Rost. V. A Shepherd and A E. Ashford plexes in an endosomal compartment (Mellman ef a/., 1986), and it might be expected that the tubules and smaller vacuoles are the endosomes, rather than the vacuoles measured. Both sets of pH values are higher than those obtained for mammalian cells where Iysosomes are reported to be pH 4'6-5'0 and endosomes ~ pH 5'5 (see Mellman ef al., 1986). Endocytosis has not been proven definitively in P. fincforius,nor in any other fungus, but the vacuole and tubule system exhibits behaviour very much like endosomes or tubular Iysosomes as seen in mammalian tissue culture cells (Shepherd ef al., 1993 a; Ashford & Orlovich, 1994). It is noteworthy that the tubule and vacuole system is most active in the terminal cells, which would be the most likely of all the cells to be actively involved in endocytosis, and that the modal pH of vacuoles in these cells is in the more alkaline range. In contrast, the system is less mobile and larger vacuoles tend to predominate in the penultimate cells, which show the more acid vacuole population. Makarow & Nevalainen (1987) also suggested that their two types of vacuole may represent two compartments in the endocytotic system, but they suggested that the more acid compartments were the endosomes. Some doubt has been cast on their data, because of the impurities likely to be present in the FITCdextran (Preston ef al., 1987). Similar pleiomorphic vacuolar networks to that of P. fincforiushave been demonstrated in actively growing hyphal tips of a wide range of other fungi (Rees, Shepherd & Ashford, 1994) and it is clear that, at least in filamentous fungi, we can no longer consider fungal vacuoles as individuaL isolated organelles with a similar composition and pH. Rather, we should consider them as a composite series of interconnected compartments that may exhibit a wide range of pH values, possibly variable, but which are not acidified to the same extent as animal Iysosomes. The research was supported by grants from the National Health and Medical Research Council (equipment) to F. W. D. R. and from the Australian Research Council to AE.A We thank W. G. Allaway for comments and criticisms of the manuscript.
REFERENCES Ashford, A E. & Orlovich, D. A (1994). Vacuole transport, phosphorus, and endosomes in the growing tips of fungal hyphae. In Pollen-Pistil Interactions. Current Topics in Plant Physiology (ed. A G. Stephenson, T-h Kao). American Society of Plant Physiologists, Rockville, U.S.A (in the press). Ashford, A E., Ryde, S. & Barrow, K. D. (1994). Demonstration of a short chain polyphosphate in Pisolithus tinctorius and the implications for phosphorus transport. New Phytologist 126, 239-247. Borst-Pauwels, G. W. F. H. (1981). Jon transport in yeast. Biochimica et Biophysica Acta 650, 8&-127. Bowman, B. J. & Bowman, E. J. (1986). H+-ATPases from mitochondria, plasma membranes, and vacuoles of fungal cells. Journal of Membrane Biology 94,83-97. Bright, G. R., Fisher. G. W., Rogowska, J. & Taylor, D. L. (1989). Fluorescence ratio imaging microscopy. In Methods in Cell Biology, Vol. 30. Fluorescence Microscopy of Living Cells in Culture. Part B. Quantitative Fluorescence
(Accepted 30 September 1994)
553 Microscopy - Imaging and Spedroscopy (ed. D. L. Taylor & Y. L. Wang), pp. 157-192. Academic Press, Inc., San Diego, CA. Cole, L., Coleman,)., Keams, A., Morgan, G. & Hawes. C. (1991). The organic anion transport inhibitor, probenecid, inhibits the transport of lucifer yellow at the plasma membrane and the tonoplast in suspension-cultured plant cells. Journal of Cell Science 99, 545-555. Davies, J. M., Brownlee, C. & Jennings, D. H. (1990). Measurement of intracellular pH in fungal hyphae using BCECF and digital imaging microscopy. Evidence for a primary proton pump in the plasmalemma of a marine fungus. Journal of Cell Science 96, 731-736. Goodall, H. & Johnson, M. H. (1982). Use of carboxyfluorescein diacetate to study formation of permeable channels between mouse blastomeres. Nature 295, 524-526. Goodwin, P. B. (1983). Molecular size limit for movement in the symplast of the Elodea leaf. Planta 157, 124-130. Grenville, D. J., Peterson, R. L. & Ashford, A E. (1986). Synthesis in growth pouches of mycorrhizae between Eucalyptus pilulans and several strains of Pisolithus linctorius. Australian Journal of Botany 34, 95-102. Guem, )., Felle, H., Mathieu, Y. & Kurkdjian, A (1991). Regulation of intracellular pH in plant cells. International Review of Cytology 12 7, 111-173. Klionsky, D.)., Herman, P. K. & Emr. S. D. (1990). The fungal vacuole: composition, function and biogenesis. Microbiological Reviews 54, 266-292. Makarow, M. & Nevalainen, L. T. (1987). Transport of a fluorescent macromolecule via endosomes to the vacuole in Saccharomyces cerevisiae. Journal of Cell Biology 104, 67-75. Martin, M. M. & Lindqvist, L. (1975). The pH dependence of fluorescein fluorescence. Journal of Luminescence 10, 381-390. Mellman, I., Fuchs, R. & Helenius, A (1986). Acidification of the endocytic and exocytic pathways. Annual Review of Biochemistry 55, 663-700. Orlovich, D. A & Ashford, A E. (1993). Polyphosphate granules are an artefact of specimen preparation in the ectomycorrhizal fungus Pisolilhus tinctorius. Protoplasma 173,91-102. Preston, R. A, Murphy, R. F. & Jones, E. W. (1987). Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran. Journal of Cell Biology 105. 1981-1987. Preston, R. A, Murphy, R. F. & Jones, E. W. (1989). Assay of vacuolar pH in yeast and identification of acidification-defective mutants. Proceedings of the National Academy of Sciences U.SA. 86, 7027-7031. Rees, B., Shepherd, V. A & Ashford, A. E. (1994). Presence of a motile tubular vacuole system in different phyla of fungi. Mycological Research 98, 985-992. Robinson, D. G. & Hillmer, S. (1990). Endocytosis in plants. Physiologia Plantarum 79, 96-104. Rost, F. W. D. (1991). Quantitative fluorescence microscopy. Cambridge University Press: Cambridge, U.K. Rothman, J. H., Yamashiro, C. T, Raymond, C. T, Kane, P. M. & Stevens, T H. (1989). Acidification of the lysosome-like vacuole and vacuolar H+ ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins. Joumal of Cell Biology 109, 93-100. Shepherd, V. A, Orlovich, D. A & Ashford, A E. (1993 a). A dynamic continuum of pleiomorphic tubules and vacuoles in growing hyphae of a fungus. Journal of Cell Science 104, 495-507. Shepherd, V. A, Orlovich, D. A. & Ashford, A. E. (1993 b). Cell-to-cell transport via motile tubules in growing hyphae of a fungus. Journal of Cell Science 105. 1173-1178. Tooze, ). & Hollinshead, M. (1991). Tubular early endosomal networks in AtT20 and other cells. Journal of Cell Biology 115, 635-653. Tsien, R. Y. (1989). Fluorescent indicators of ion concentrations. In Methods in Cell Biology, Vol. 30. Fluorescence Microscopy of Living Cells in Culture. Part B. Quantitative Fluorescence Microscopy - Imaging and Spectroscopy (ed. D. L. Taylor & Y. L. Wang), pp. 127-156. Yamashiro, C. T, Kane, P. M., Wolczyk, D. F., Preston, R. A & Stevens, T H. (1990). Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar translocating ATPase. Molecular and Cellular Biology 10, 3737-3749.