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

JULIA M. DAVIES1'3'*, C. BROWNLEE2 and D. H. JENNINGS1

1 Department of Genetics and Microbiology, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK^Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK^Present address: Department of Biology, University of York, Heslington, York Y01 5DD, UK

* Author for correspondence

Summary

The facultative marine fungus, Dendryphiella salina,has the most negative membrane potential yetrecorded for a marine organism. The ionic basis forthis is thought to be through the action of a primaryproton pump, though there exists the possibility ofelectrogenic pumping of Na+ or Cl~, given the highambient concentration of these ions. Fluorescenceratio imaging microscopy with the pH-sensitive flu-orescent probe 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) has been used to esti-mate intracellular pH. Hyphae loaded readily withBCECF after incubation with the acetoxymethylester (BCECF/AM). Mean resting intracellular pH(pHi) was 7.3, calculated by comparing 490/450 nmfluorescence ratios with in vivo calibration curves

Introduction

Mesophytic plants and fungi energise secondary solutetransport through vectorial translocation of H+ at theplasma membrane (Serrano, 1984). The active extrusion ofH+ generates a proton electrochemical gradient (A^H+)that can be partitioned into membrane potential (AV) andpH (ApH) components. Solute uptake is coupled to the re-entry of H+. For a marine fungus, the high ambient pH(pH 8) could put H+ at a premium and necessitate the useof Na+ or Cl~ as the working ion of primary and/orsecondary systems. Precedents for electrogenic primaryNa+ and Cl~ transport come from the marine algaeHalicystis (Blount and Levendahl, 1960) and Acetabularia(Saddler, 1970). Dendryphiella salina is a facultativemarine fungus and is thought to have evolved from aterrestrial ancestor (Kohlmeyer, 1974; Kohlmeyer andKohlmeyer, 1979). The possible retention of typical meso-phytic H+-based transport systems has been explored in aseries of electrophysiological studies (Brownlee, 1984;Davies et al. 1990). In D. salina, the large negative A'V wasfound to be metabolic in origin and sensitive to specificinhibitors of the plant and fungal plasma membrane H+-ATPase. Uptake of glucose depolarised A1!* in a mannerconsistent with the operation of a H+: solute symporter.

Further evidence for primary H+ pumping would comefrom the estimation and manipulation of the protonJournal of Cell Science 96, 731-736 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

obtained by pH equilibration using nigericin. Dis-tinct pH compartments could be observed, corre-sponding to cytoplasmic and smaller vacuolar com-partments. Sodium azide reversibly reduced pHi byan average of 0.51 of a pH unit, though the responsevaried between individual hyphae. Inhibiting theplasmalemma ATPase with orthovanadate also re-versibly decreased pH|. The results support the pres-ence of a proton pump in the plasmamembrane. Theenergetic and evolutionary implications are dis-cussed.

Key words: intracellular pH, ratio imaging, proton pump,marine fungus.

electrochemical gradient (A/iH+). The morphology of D.salina renders the use of H+-sensitive microelectrodesunsuitable and the magnitude of Aty might distort pHestimates using lipophilic probes (Raven and Smith, 1978).An alternative method uses the fluorescent pH probeBCECF. The acetoxymethyl ester form of the dye diffusesinto the cell where esterase activity releases the pH-sensitive free acid (Tsien, 1981; Bright et al. 1987; Dixon etal. 1989; Paradiso et al. 1984).

In this work, BCECF has been used to estimate pHj andto assess the effects of azide and vanadate as inhibitors ofthe mitochondrial H+-ATPase and plasmamembraneATPase, respectively.

Materials and methods

Stock cultures of Dendryphiella salina Sutherl. (Pugh & Nicot)pp6604 were grown on plates of (w/w) 0.5% glucose, 0.3%tryptone, 1 % agar in filtered sea water for 14 days at 20°C. Then,0.6 cm discs from the periphery of cultures were inoculated onto2 cm x 2 cm cellophane squares laid on fresh agar. These wereincubated for 3 days at 20 °C.

Fluorescence microscopyHyphae growing on cellophane were bathed in lcm3 glucose-artificial sea water medium (GASW: (in mM) NaCl, 500; KC1, 10;

731

CaCl2, 10; KHCO3, 2.5; Tris/HCl, 10; pH8.0). BCECF/AM wasadded to the bathing medium to a final concentration of 6 fiM froma 1 mM stock solution in dimethyl sulphoxide. Hyphae wereincubated for 60-90 min and then washed with 10 x 1 cm3 portionsof GASW. Fluorescence was observed with a modified Zeissfluorescence microscope with a x40 Neofluar water immersionobjective. Excitation wavelengths were produced with a SOWmercury vapour lamp and 10 nm bandwidth filters (490 and460 nm). Filters were changed manually. Excitation was restric-ted to ~ 10 s to minimise dye bleaching. Fluorescence was moni-tored at 530 nm through a 50 nm bandwidth filter. Images wererecorded with an image-intensified CCTV camera (Panasonic,Japan) and stored on video tape.

Image analysisImages were digitised using a Kontron digital image analyserwith IBAS software (512x512 pixels, 256 grey levels). Aftersubtraction of autofluorescence, recorded before dye loading,490/450 nm ratio images of dye-loaded cells were obtained (Brightet al. 1987). Up to 10 frames were averaged for each image.Brightness profiles across selected hyphal transects in the ratioimages were compared with calibration curves (see below) to giveintracellular pH values.

CalibrationAn in vitro calibration curve was obtained from drops of buffer(130 mM KC1, lmM MgCl2, 15 mM Mes, 15 mM Hepes, pH5-9)(Bright et al. 1987) on a microscope slide. In situ calibration wascarried out on dye-loaded hyphae after incubating in either lowK+/nigericin (GASW+15mM Mes, 15 mM Taps, 3mM sodiumazide, with 10//gcm~3 nigericin, pH5-9) or high K+/nigericin(the above solution modified by substituting 100 mM NaCl with100 mM KC1). After equilibration in these solutions for 5-10 min,fluorescence was recorded and hyphae were washed with 5x1 cmbuffer at the next pH. The sequence was repeated for each pHtested. Calibration curves (fluorescence ratio as a function of pH)were constructed from these treatments for comparison withexperimental pH measurements.

Results

After loading with BCECF/AM, fluorescence was restric-ted to viable hyphae. Conidia or hyphae that had beendeliberately damaged did not fluoresce. BCECF/AM hadno effect on hyphal extension rates (not shown). Dyedistribution was generally diffuse throughout the cyto-plasm, though occasional bright spots were observed,probably representing accumulation into small vacuoles.Such regions were avoided in quantitative measurementsof cytoplasmic pH where possible.

Experimental and calibration ratio values were com-puted by averaging 10-20 points along a profile of theratio image. All three calibration curves showed linearityin the pH range 6-8 (Fig. 1). The in vitro and highK+/nigericin in situ calibrations showed good agreementbut the low K+/nigericin in situ standards gave signifi-cantly lower mean ratio values. Fig. 2A-C shows bright-field, 490 nm and 450 nm fluorescence images of a BCECF-loaded hypha bathed in GASW, pH8.0. The corresponding490/450 nm ratio image is shown in Fig. 2D. The highbackground ratio is due to slight persistence of extracellu-lar fluorescence following washing, possibly due to adsorp-tion of leaked dye to the cellophane substrate or incom-plete washing of the BCECF/AM used during loading.Fluorescence from out-of-focus hyphae will also contributeto the background ratio. The high extracellular ratio valuedoes not significantly affect the hyphal ratio values, sincethe absolute fluorescence is much higher from the loadedhyphae. For clearer presentation of the hyphal ratio image

2.5

•S 1-5

0.5

5 6 7 8 9PH

Fig. 1. In vitro (A) and in situ ( • , • ) pH calibration curves;(•) hyphae equilibrated with 112.5 mM K+/nigericin solutions;(•) hyphae equilibrated with 10 mM K+/nigericin solutions.

Fig. 2. A. Bright-field image of mature hyphae. Bar, 10 /an.B,C. 490 and 450 nm fluorescence images of the same BCECF-loaded hyphae in GASW. D. 490/450 nm ratio image.

and to define precisely the hyphal edges, the bright-fieldimage of the hypha has been used as a template to maskthe background (Fig. 3A). The hypha shows a complexdistribution of pHj values. A mean pH^ was calculatedfrom 15 profile points (Fig. 3A) using the high K+ cali-bration. The mean intracellular pH from 10 hyphaebathed in GASW at pH8 was 7.33 (±0.34) (ire vitrocalibration) or 7.30 (±0.32) (high K+ calibration). Theseare gross estimates, which do not distinguish betweencytoplasmic and the smaller vacuolar compartments. Tworatio profiles across clearly vacuolar regions (Fig. 3A,arrows) gave a mean vacuolar pH of 5.85. A 5 minincubation with 3mM sodium azide produced a clearreduction in fluorescence ratio (Fig. 3B). The pH of thisazide-treated hypha was estimated to be <5.5 Somespatial variation of the ratio persisted. Washing withGASW followed by a 5 min recovery resulted in a pH

732 J. M. Davies et al.

6Hypha Hypha Hypha

100 200 300Points b

0 r

Hypha Hypha Hypha

0 100 200 300

Points

2-

Hypha Hypha Hypha

0 100 200 300Points

Fig. 3. Same ratio image Withbackground ratio masked using thebright-field image as a template todelineate the hyphal edge. The brokenline indicates profile and the directionof the transect (a-b). Low pH vacuolarregions are indicated by separatenumbered arrows. The correspondingbrightness profile along the transects isalso shown. Arrows on the profilecorrespond to the low pH regions alongthe transect. B. Ratio image andcorresponding brightness profile of thesame hyphae treated with sodium azidefor 5 min. C. Ratio image andbrightness profile after washing withGASW.

Table 1. The effect of 3 mM sodium azide on pHt

Pre-azide +Azide GASW-washed pH decrease

6.950.32

6.540.49

7.20.39

0.410.36

Results represent mean values from four separate experiments. pHvalues were computed from high K+/nigericin calibration curves.

increase to 6.62±0.22 (Fig. 3C). The pooled results fromfour representative experiments are shown in Table 1. ThepH, decrease after azide treatment ranged from 0.1 to1.67 pH units. In all but one hypha (which showed no

response) a decrease in pH, was observed. A mean pHdecrease of 0.41 (±0.4) was calculated.

Fig. 4A shows a ratio image of mature hyphae in GASWprior to treatment with vanadate. This image did not showa high background ratio except for regions close to thehyphae, probably due to more effective washing of thepreparation. A mean pH of 7.44 (±0.23) was calculatedacross the profile shown (Fig. 4A). A 5 min exposure to3 mM vanadate lowered the pH to 5.83(±0.19) (Fig. 4B). AGASW wash and 5 min recovery returned pH to 91 % of itsoriginal value (6.78 (±0.31); Fig. 4C). The results fromfour separate vanadate experiments are shown in Table 2.At 3mM, vanadate produced a mean pH decrease of 1.35unite. The effect was almost fully reversible.

pHt in marine fungal hyphae 733

6-

0J

100 2ooPoints

6-

100Points

200

4-

100Points

200

Fig. 4. A. Ratio image andbrightness profile along thetransect indicated of BCECF-loaded hyphae in GASW.B. Ratio image andcorresponding profile aftertreatment with 3 mM sodiumorthovanadate for 5 min. C. Afterwashing with GASW. Bar, 10 /on.

Table 2. The effect of 3 mM sodium orthovanadate on

Pre-vanadate +Vanadate GASW-washed pH decrease

7.680.31

6.170.29

7.820.55

1.350.33

Results represent means of four separate experiments.

Discussion

Measurements of pHL in plants are scarce. The accuracy ofthose presented here depends primarily on the reliability

of the calibration. In situ calibration compensates directlyfor spectral artefacts due to binding or metabolism of thedye. The use of nigericin in this context is well establishedand dye leakage rate does not appear to be promoted bythis ionophore (Chaillet and Boron, 1985). The calibrationcurves made with nigericin clearly show the dependence ofclamped pH on external K+. Nigericin promotes pHequilibration through the exchange of K; for HJ". Atequilibrium, [K+]i/[K+]0=[H+]i/[H+]0. The_rK+]; of D.salina has been estimated to be 114mMkg cell water(Galpin et al. 1978) and only the use of high external K+

(112.5 mM) produced ratio values similar to that producedby the free dye in vitro. The sensitivity range reported hereagrees well with that reported in animal cells (Kink et al.

734 J. M. Davies et al.

1982). Intracellular pH measurements from the marinealga Chaetomorpha have revealed that the cytoplasm ismaintained slightly alkaline to sea water at pH 8-8.3(Raven and Smith, 1980), thermodynamically consistentwith the action of a primary proton pump during theformation of ApH. In contrast, the average pH; of D. salinaappears to be maintained at a value near neutral, similarto that found in terrestrial algae and higher plants. Moststriking is the similarity between D. salina and theterrestrial fungus Neurosporu crassa, which maintainspH| at approximately 7.2 when pH,, is 5.8 (Sanders andSlayman, 1982). Both fungi displayed a range of pH;values, possibly reflecting the different physiological agesof different hyphae tested. The values reported here mayalso vary according to the degree of vacuolation of aparticular hypha. The vacuolar pH estimate for D. salinaresults from a composite of signals from vacuoles and thesurrounding layer of cytoplasm. However, the result isconsistent with an acidic vacuolar compartment, typical ofalgae and plants, suggesting an inwardly directed protonpump for tonoplast energization.

Inhibition of primary H+-pumping should induce atleast transient intracellular acidification. The acidifi-cation produced by azide can be interpreted as inhibitionof such pumping via the mitochondrial ATP supply. Thereversibility of the response (90 %) compares well with themean recovery of A^ (93 %) after treatment with the sameconcentration of azide (Davies et al. 1990). Azide-inducedacidification cannot be explained by the dissociation of thisweak acid (pK& 4.74) or by an uncompensated H+-leakcurrent. If acidification were the result of dissociation,then AV should hyperpolarise as decreased pH( acceler-ates proton pumping (Sanders and Slayman, 1982). In-stead, azide-induced depolarisations have been observed,with A*V decreasing to — 50 mV with 3mM azide (Brown-lee, 1984; Davies et al. 1990). If A^H+ is calculated forconditions of azide inhibition, substituting AvI/=-50mV,pHo8.0, pHj6.8, where A/iH+=*FAV+2.303flT (pH,,-pHj), z is the valency of H+ and F (Faraday constant), R(the gas constant) and T (absolute temperature) have theirusual values; a net outward driving force for H+ isindicated (=20 mV). Instead, it seems reasonable that theH+ load is metabolic in origin.

Intracellular acidification and AW depolarization (to-60 mV) by 3mM vanadate are both fully reversible(Davies et al. 1990). Again, acidification cannot beexplained by dissociation (pKa 8) or by a H+ leak as A;tH+

is equivalent to 70 mV under these conditions. As arelatively specific inhibitor of the plasma membrane H+-ATPase, vanadate-induced acidification indicates the pres-ence of a primary H+ pump in D. salina.

Implications for the retention of a H+ pump in a marineorganism may be assessed by calculating A/*H+. For D.salina, this is approximately -20kJmol~1 (where AXV=—250 mV and ApH=0.7unit). This is consistent with theA/*H+ of mesophytic plants, —20 to — 25kJmol~1 (Smithand Raven, 1979), which is sufficient to drive secondaryuptake of solutes. Raven and Smith (1980) postulated thatthe pH, of marine plants would be greater than pH 8 tocreate a sufficiently large A/*H+. In retaining a mesophy-tic near-neutral cytoplasmic pH, D. salina may overcomethe environmental constraint of high ambient pH bymaintaining a large negative A 4* to bring AjtH+ to ausable level.

At —250 mV, A*P of D. salina is the largest yet recordedfor a marine organism. Such an interpretation, however,assumes that H+-coupled solute uptake systems are

mechanistically able to use AV when ApH has the wrongpolarity. A comparison may be made with alkalophilicbacteria that maintain relatively acidic cytoplasm in highalkaline habitats (pH,-, 10-11). For such organisms, it ispossible that A*P is sufficiently negative to generate asuitable AjtH+ (Krulwich and Guffanti, 1983).

There remains the possibility that the putative H+

pump does not act in isolation but co-exists with primaryNa+ and/or Cl~ pumps. However, previous electrophysio-logical experiments have shown that substitution of exter-nal Na+ and Cl~ has little or no effect on A1!' (Davies et al.1990). In contrast, changes in pH<, produced significantshifts. Alkalinization of the medium was observed oninhibiting primary transport or initiating sugar co-trans-port. Such results suggest that the H+-ATPase is the soleelectrogenic pump at the plasmalemma.

Overall, it would appear that the successful colonisationof the sea by the putative terrestrial ancestor of D. salinadid not require or produce major changes in the primarytransport system. The main adaptation would be in themechanism of secondary H+-coupled systems to functionwith reversed ApH polarity. Under the severe selectiveconditions imposed by the sea, even the appearance of asubtle or relatively inefficient adaptation could enable anorganism to colonise and radiate.

The authors thank the Science and Engineering ResearchCouncil of the United Kingdom for financial support.

References

BLOUNT, R. W. AND LEVENDAHL, B. H. (1960). Active sodium and chloridetransport in the single celled marine alga Halicystis ovalis. Actaphysiol. scand. 49, 1-9.

BRIGHT, G. R , FISCHER, G. W., ROGOWSKA, J. AND LANSINQ-TAYLOR, D.(1987). Fluorescence ratio imaging microscopy. Temporal and spatialmeasurements of cytoplasmic pH. J. Cell Bwl. 104, 1019-1033.

BEOWNLBE, C. (1984). Membrane potential components of the marinefungus Dendryphiella salina (Suth). Nicot & Pugh. Possibleinvolvement of calmodulin in electrophysiology and growth. NewPhytol. 97, 15-23

CHAJIXKT, J. R. AND BORON, W. F. (1985). Intracellular calibration of anpH-sensitive dye in isolated, perfused salamander proximal tubulea. J.gen. Phyaiol. 86, 766-794.

DAVIES, J. M., BROWNLEE, C. AND JENNINGS, D. H. (1990).Electrophyaiological evidence for an electrogenic proton pump and theproton symport of glucose in the marine fungus Dendryphiella salina.J. exp. Bot. 41, 449-466.

DIXON, G. K., BROWNLBK, C. AND MERRETT, M. J. (1989). Measurement ofinternal pH in the coccolithophore Emiliania huxleyi usingBCECF/AM and digital imaging microscopy. Planta 178, 443-449.

GALPIN, M. F. J., JENNINGS, D. H., OATES, K. AND HOBOT, J. A. (1978).Localization by X-ray microanalysis of soluble ions, particularlypotassium and sodium in fungal hyphae. Expl Mycol. 2, 258-269.

KOHLMBYER, J. (1974). On the definition and taxonomy of higher marinefungi. VerOff. Inst Meeresforsch. Bremerh. Supplement 5, 263-286.

KOHLMEYER, J. AND KOHLMBYBR, E. (1979). Marine Mycology: The HigherFungi Academic Press, New York, San Fransisco, London.

KRULWICH, T. A. AND GUPFANTI, A A. (1983). Physiology of acidophilicand alkalophilic bacteria. Adv. microb. Physiol. 24, 173-213.

PARADISO, A. M., TSIEN, R. Y. AND MACHEN, T. E. (1984). Na+/H+

exchange in gastric glands as measured with a cytoplasmically-trapped, fluorescent pH indicator. Proc natn. Acad. Sci. U.S-A. 81,7436-7440.

RAVEN, J. A. AND SMITH, F. A. (1978) Effect of temperature and externalpH on the cytoplasmic pH of Chara corallina. J. exp. Bot. 29, 863-866.

RAVEN, J. A. AND SMITH, F. A. (1980). Intracellular pH regulation in thegiant celled marine alga Chaetomorpha darwinii. J. exp. Bot. 31,1357-1369.

RINK, T. J., TSIBN, R. Y. AND POZZAN, T. (1982). Cytoplasmic pH and freeMg2* in lymphocytes. J. Cell Biol. 9S, 189-196.

SADDLER, H. D. W. (1970). The membrane potential of Acetabulariamediterranea. J. gen. Physiol. 55, 802-821.

SANDIRS, D. AND SLAYMAN, C. L. (1982). Control of intracellular pH.Predominant role of oxidative metabolism, not proton transport, in the

pHl in marine fungal hyphae 735

eukaryotic microorganism Neurospora crassa. J. gen. Physiol. 80, Intracellular pH measurements in Ehrlich ascites tumor cells utilizing377-402. 8pectroscopic probes generated in situ. Biochemistry 18, 2210-2218.

SERRANO, R. (1984) Plasma membrane ATPase of planta and fungi as a TSIEN, R. Y. (1981). A non-destructive technique for loading calciumnovel type of proton pump. In Current Topics in Cell Regulation, vol. buffers and indicators into cells. Nature 290, 527-528.23, pp. 87-126. Academic Press, New York and London.

SMITH, F. A. AND RAVEN, J. A. (1979). Intracellular pH and itsregulation. A. Rev PI. Physiol. 30, 289-311.

THOMAS, J. A., BUSCHBAUM, R. N., ZIMNIAK, A. AND RACKER, E. (1979). (.Received 17 April 1990 - Accepted 10 May 1990)

736 J. M. Davies et al.