2845Journal of Cell Science 110, 2845-2853 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS3638

Intracellular Ca 2+ signals in Dictyostelium chemotaxis are mediated

exclusively by Ca 2+ influx

T. Nebl and P. R. Fisher

School of Microbiology, La Trobe University, Bundoora, VIC 3083, Australia*Author for correspondence (e-mail: fisher@lumi.latrobe.edu.au)

We measured folate- and cAMP-induced changes incytoplasmic free calcium concentration ([Ca2+]i) usingrecombinant aequorin reconstituted in livingDictyostelium cells with coelenterazine-h. The resultingsemi-synthetic protein displayed increased sensitivity toCa2+ allowing accurate measurement of chemo-attractant-induced transients at low resting levels. Bothfolate- and cAMP-induced Ca2+ responses were develop-mentally regulated, exhibited remarkably similarkinetics and were dependent on the relative rather thanthe absolute magnitude of increases in attractant con-centration. They began after a short delay of 5-10seconds, leading to a maximum increase in cytosoliccalcium concentration after ~25 seconds and a return tobasal level within ~60 seconds after stimulation.Responses elicited by the two chemoattractants weredose-dependent and saturated between 4 and 20 µM.They depended on the presence of free extracellular

calcium ions and were inhibited in a concentration-dependent manner between 10−4 and 10−5 M. In accor-dance with 45Ca2+-uptake measurements by Milne andCoukell (J. Cell Biol. (1991) 112, 103-110), both responseswere also completely inhibited by 15µM Ruthenium Red,15 µM carbonylcyanide m-chlorophenyl-hydrazone(CCCP) and 500µM gadolinium ions. Under conditionsthat prohibited influx of Ca2+ from the extracellularmedium there were no detectable changes in [Ca2+]i thatcould be related to a separate release of the ion fromintracellular stores. Together, these results show that theCa2+ signals involved in chemotaxis correlate temporallywith actin depolymerization (not polymerization) and aremediated by Ca2+ influx, not IP3-mediated intracellularrelease.

Key words:Dictyostelium discoideum, Calcium, Signal transduction,Chemotaxis

SUMMARY

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INTRODUCTION

The cellular slime mould Dictyostelium discoideumprovidesan attractive eukaryotic model for studying the molecular baof reception and transduction of chemoattractant signaChemotactic processes are vital to the growth and developmof this organism. Growing amoebae can sense folic asecreted by bacteria, enabling them to track down thmicrobial food source. After a few hours of starvation Dic-tyosteliumcells develop a receptor-mediated signaling systebased on extracellular cAMP. Periodic pulses of cAMemanating from aggregation centres organize the accumulaof about 105 solitary amoebae into a multicellular structurewhich subsequently undergoes differentiation and eventuadevelops into a fruiting body consisting of spores and stcells.

The signal transduction pathways involved in chemotaand differentiation of Dictyosteliumare believed to be highlyhomologous to those of higher eukaryotic cells (reviewed Van Haastert et al., 1991). Binding of folate and cAMP at cocentrations in the nanomolar range to specific cell surfachemoreceptors has been reported to rapidly activate phpholipase C and guanylate cyclase via a G-protein-dependpathway, resulting in increased intracellular inositol-1,4,

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trisphosphate (IP3) and guanosine 3′,5′-cyclic monophosphate(cGMP) concentrations within seconds (Mato et al., 1977Wurster et al., 1977; Europe-Finner and Newell, 1987; VaHaastert et al., 1989). Chemotactic excitation also triggerstransient influx of Ca2+ from the extracellular medium (Wicket al., 1978; Bumann et al., 1984; Milne and Coukell, 1991Menz et al., 1991), followed by Ca2+ extrusion and sequestra-tion via membrane associated Ca2+-ATPases (Böhme et al.,1987; Rooney et al., 1994). The second messengers activdirectly or indirectly, the physiological response. Cytoskeletaprocesses involved in chemotactic migration include locallrestricted changes in the amount of filamentous actin (reviewby Schleicher and Noegel, 1992; Noegel and Luna, 1995) athe phosphorylation, assembly and association of myosin with the actin cytoskeleton (reviewed by Newell, 1995).

Direct demonstration of the role of cytosolic free calciumconcentrations during these processes requires the measment of temporal and local changes in [Ca2+]i in intact Dic-tyostelium cells. The chemoattractant-induced Ca2+ uptakeshould lead to an increased [Ca2+]i in the micromolar range(Bumann et al., 1984), but until now it was unclear whethethere is, in fact, any significant elevation of cytoplasmic Ca2+

levels in response to chemotactic stimuli. The first direcevidence for a transient increase in [Ca2+]i following stimula-

2846

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tion of Dictyosteliumcells with chemoattractants at physiological (nanomolar) concentrations was provided by Abe et(1988) using Fura-2 introduced into Dictyosteliumcells byelectroporation. Others, however, found that this loadiprocedure caused cell damage, and did not detect glochanges in [Ca2+]i in response to submicromolar concentrations of cAMP after scrape-loading single cells with dextracoupled fura-2 (Schlatterer et al., 1992, 1994). In fact it hbeen suggested that [Ca2+]i may be so strongly buffered thaCa2+ uptake in response to physiological attractant stimuli donot result in any measurable increases in global cytosolic f[Ca2+]i. If true this would make physiologically significanroles for [Ca2+]i in chemotaxis highly unlikely. Recently,Yumura et al. (1996) were able to confirm the original findinby Abe et al. (1988), using fura-2 coupled to bovine serualbumin introduced into intact Dictyostelium cells by amodified electroporation procedure. However, because thoccurred well before the observed uptake of 45Ca2+, thereported responses are not readily attributable to Ca2+ influx,despite being dependent on extracellular Ca2+ (Yumura et al.,1996). The contradictions between the various results mayexplained by the different loading methods and reflect tvarious technical difficulties encountered with the use of florescent Ca2+-sensitive dyes in Dictyostelium. In an attempt toovercome these problems Saran et al. (1994) introducenovel, non-perturbing method for monitoring [Ca2+]i in Dic-tyosteliumbased on the recombinant photoprotein aequorThis method has facilitated the study of patterns of high, fr[Ca2+]i at multicellular stages of Dictyostelium(Cubitt et al.,1995), but its usefulness for monitoring [Ca2+]i responses tocAMP has been impeded by the relatively low Ca2+-affinity ofnative aequorin. In this work we used a semi-syntheaequorin with improved light emission properties (Shimomuet al., 1989) and demonstrate its suitability for measuring smchanges in [Ca2+]i at the very low basal levels of free ion founin Dictyosteliumamoebae.

The main focus of the present work was to accuratemeasure the temporal change of [Ca2+]i in response tochemoattractants, to analyse their requirement for extracelar Ca2+, and to investigate whether chemoattractants causdetectable release of Ca2+ from intracellular stores under con-ditions that block or inhibit Ca2+ influx. The results provide forthe first time a clear and accurate picture of the changes[Ca2+]i elicited by physiological chemoattractant stimuli. It habeen proposed that one signal transduction pathway involin chemotaxis proceeds from receptor and G-protein activatvia IP3-mediated Ca2+ release to actin polymerization (Newelet al., 1990). The findings here indicate that chemoattractaelicit changes in [Ca2+]i only by means of Ca2+ influx, with nocontribution from Ca2+-independent, IP3-mediated intracellu-lar release. Furthermore, the [Ca2+]i responses to chemotacticstimuli are shown to correlate not with actin polymerizatiobut depolymerization. This knowledge should facilitatreassessment and better understanding of the roles of [C2+]iin mediating chemotaxis.

MATERIALS AND METHODS

Materials1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-1,4-tetraacetic acid

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(BAPTA), Calcium Calibration Buffer Kit with 1 mM free Mg2+,coelenterazine, coelenterazine-h and Pluronic F-127 were purchfrom Molecular Probes, Inc. (Eugene, OR 97402-0469 UScAMP, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP)EGTA, folic acid, GdCl3 and Ruthenium Red were purchased froSigma-Aldrich Pty., Ltd (Castle Hill, NSW 2154 Australia), whil0.1 M CaCl2 Calibration Standard was purchased from ATI Orio(Boston, MA 02129 USA).

Construction of a D. discoideum expression plasmidencoding the apoaequorin geneThe apoaequorin-coding region from cDNA clone pAQ2 (Knight al., 1991), kindly provided by Mark R. Knight (University oEdinburgh, UK), was amplified by the polymerase chain reactusing 5′-phosphorylated primers containing terminal restrictioendonuclease recognition sites to permit efficient cloning. An aaequorin expression plasmid was constructed by insertion of a 0.PstI/XhoI PCR product encoding the entire apoaequorin cDNA inPstI/XhoI cut Dictyosteliumplasmid pDNeo2. The resulting construcpPROF120, contains a gene fusion between the first 8 codons oDictyosteliumactin 6 gene and the apoaequorin gene. Expressiontermination of the actin6-apoaequorin fusion protein are regulatedthe Dictyosteliumactin 6 promoter and actin 8 transcription termintor, respectively.

Transformation and cell cultureCells of the Dictyosteliumwild-type strain AX2 were transformedwith 15 µg pPROF120 DNA according to a standard Ca2+-phosphateprocedure (Nellen et al., 1984) and selected on M. luteuslawns onSM-agar containing 15 µg/ml G418 as previously described (Wilczynska and Fisher, 1994). Transformants were purified by streak cloon M. luteus lawns on SM-agar containing 20 µg/ml G418. Pureclones were transferred to 24-well culture plates containing Hgrowth medium supplemented with antibiotics (10 µg/ml strepto-mycin, 50 µg/ml ampicillin and 20 µg/ml G418). Transformants werescreened for high expression by Northern blot analysis of total Rusing a DIG-labelled aequorin cDNA probe. One of the cell linexpressing the highest amount of aequorin (HPF275) was used fosubsequent in vivo studies.

Partial purification of aequorin and calibration ofluminescent signalsIn order to assay aequorin activity we prepared lysates of cexpressing recombinant apoaequorin, incubated them in the presof a large excess of synthetic coelenterazine to generate activetoprotein and subjected the resulting reaction mixture to gel filtratto obtain partially purified aequorin. To do this vegetative cells groin HL5 medium were harvested at a density of 4×106 cells/ml, washedtwice and resuspended in two volumes of lysis buffer (10 mM Pip30% (w/v) sucrose, 40 mM sodium pyrophosphate, 10 mM EDTpH 6.9) containing a mixture of protease inhibitors (30 µg/ml PMSF,5 µg/ml leupeptin and 5 µg/ml pepstatin A). Cell extracts wereprepared by repeated cycles of freeze-thawing and centrifuged fominutes at 10,000 g to sediment the debris. The incorporation reactiwas started by addition of 5 mM β-mercaptoethanol and 100µM ofcoelenterazine (or coelenterazine-h) and was incubated overnig4°C. The reaction mixture was then subjected to ammonium sufractionation and gel filtration on Sephadex G-50 according to method of Blinks et al. (1978). The fractions containing the highphotoprotein activity were concentrated by hydrophobic adsorptionphenyl-Sepharose CL-4B and desalted by gel filtration throuSephadex G-25 by the method of Shimomura (1991). All the absteps were carried out at 4°C and all buffers were prepared fdeionized water and contained EDTA to minimize the dischargeaequorin. For the determination of calcium concentration-effcurves 25 µl aliquots of the active photoprotein were injected into 2ml volumes of 10 mM K2EGTA/CaEGTA buffers covering the rang

2847Intracellular Ca2+ responses in Dictyostelium chemotaxis

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of 0 to 40 µM free [Ca2+] in 100 mM KCl, 10 mM MOPS, pH 7.2,and 1 mM free [Mg2+] (Calcium Calibration Buffer Kit with 1 mMfree Mg2+). Light emission was measured with a microcomputebased photometric system as previously (Schaap et al., 1996). Cbration curves for apoaequorin reconstituted with normal coelenazine and coelenterazine-h were drawn relating the fractional rataequorin consumption to [Ca2+].

In vivo aequorin reconstitution and measurement of[Ca2+]i

Generally, in vivo reconstitution of the Ca2+-sensitive photoprotein wasachieved by incubating in the presence of 2.5 µM-5 µM coelenter-azine-h 1-2×107 cells/ml transformed cells suspended in 50 ml growmedium shaken at 120 rpm at 22°C for >6 hours. Coelenterazine-hroutinely added from a 2,000-fold concentrated suspension methanol containing 20% (w/v) Pluronic F-127. For measuremenfolate-induced responses cells were grown in 500 ml HL5 to a density of ~1×106 cells/ml, centrifuged (500 g/5 minutes) andincubated in the presence of coelenterazine-h as above. In ordemeasure cAMP-induced responses cells were grown in 500 ml Hmedium to a density of ~2.5×106 cells/ml. To initiate development,cells were harvested, washed twice in 400 ml cold PBS, once in c400 ml MES development buffer (MES-DB: 10 mM MES/NaOH, p6.2, 10 mM KCl, 0.25 mM CaCl2) and incubated in the presence ocoelenterazine-h at 2×107cells/ml in 50 ml MES-DB for >7 hours.Prior to in vivo measurement 5 ml aliquots of the cell suspension wwithdrawn, collected at 500 g for 1 minute and gently washed twicebefore resuspension in 5 ml MES-DB. To investigate the effectknown inhibitors of chemoattractant-induced Ca2+ uptake, theinhibitors were added at concentrations indicated in the Figure legeapproximately 2-3 minutes prior to measurement of chemoattractinduced responses. To lower extracellular [Ca2+] to a level approach-ing ‘zero’ the bathing medium was replaced with nominally Ca2+-freeMES-DB containing 2 mM BAPTA. During all measurements cell supensions were stirred at 100 rpm in 20 ml standard assay vessels immediately in front of a low noise photomultiplier. Chemotactstimuli were delivered by injection of 25 µl of chemoattractants from200-fold concentrated stock solutions in deionized water.

Fig. 1.Measurement of [Ca2+]i in D.discoideumusing recombinant aequorin.(A) Time course of in vivo reconstitution ofrecombinant semi-synthetic aequorin.Dictyosteliumcells expressing recombinantapoaequorin were incubated at a density of1×106 cells/ml in 20 ml HL5 growth mediumcontaining 5 µM coelenterazine-h. After 0.5, 1,2, 4 and 6 hours active aequorin was dischargedby lysing 1 ml aliquots of washed cells in lysisbuffer (10 mM MOPS, 10 mM Ca-acetate, 1%Triton X-100, pH 7.0). Each time pointrepresents an average of 3 individualdeterminations. The bar graph illustrates theenhancing effect of the dispersing agentPluronic F-127 on the yield of Ca2+-sensitiveaequorin from in vivo reconstitution insuspensions of Dictyosteliumcells. Data are expressed as the perceliving cells in growth medium containing 20 µM coelenterazine-h for 6reconstitution using 5 µM coelenterazine added from 5 mM methanoPluronic F-127. Each bar represents the average of 3 individual deconsumption to free calcium ion concentration under conditions thindividual determinations using aliquots of recombinant apoaequo(n,,,e). Rate constants were determined by expressing the light saturating [Ca2+]. All determinations were made in 10 mM K2EGTA/Ca100 mM KCl, 10 mM MOPS, pH 7.2 and 1 mM free Mg2+ at 22°C as dexactly the same luminometer configuration as for this calibration

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Interpretation of light measurementsAt the end of each experiment multiple 0.5 or 1 ml aliquots of thcells were disrupted in lysis solution (10 mM MOPS, 10 mM Caacetate, 1% Triton X-100, pH 7.0), causing discharge of the aequoremaining in the preparation. This allows retrospective normalizatiof the signals recorded from the cells prior to lysis on the basis of in vitro concentration-effect curve of aequorin luminescence. Corretion of aequorin consumption throughout an experiment, as wellnormalization and conversion of in vivo signals into values of [Ca2+]iwas achieved on a microcomputer using our own purpose-desigsoftware written in the ‘S’-programming language for data analysand graphical display (Becker et al., 1988).

RESULTS

Expression and reconstitution of a semi-syntheticaequorin with increased Ca 2+-sensitivity in D.discoideumThe protein produced by transformants expressing apaequorin cDNA lacks its prosthetic group, coelenterazine, ais totally inactive alone. In vivo reconstitution of the functionaphotoprotein was obtained in a simple, non-perturbing way incubating cells in growth medium or development buffer cotaining the chromophore. The concentration necessary obtain maximal reconstitution was ~20 µM coelenterazine. Wefound that inclusion of the dispersing agent Pluronic F-12enhanced reconstitution at lower concentrations of tluminophore (Fig. 1A). In vivo reconstitution in the presencof 5 µM coelenterazine-h resulted in a sub-optimal yield (opebar, 5−) compared to reconstitution with 20µM coelenterazine-h (solid bar, 20−). Addition of 5 µM of the highly hydropho-bic chromophore from a methanolic stock solution containinPluronic-F127 (striped bar, 5+) significantly enhanced celoading. In experiments that require large volumes of cell su

ntage of reconstitution relative to the maximum yield from incubation of hours (solid bar, 20−). Shown are the relative efficiencies oflic stock with (striped bar, 5+) or without (open bar, 5−) 20% (w/v)terminations. (B) Calibration curve relating the rate constant for aequorinought to be appropriate to the cytoplasm of amoebae. Points on the curve arerin reconstituted with standard coelenterazine (h,s), or coelenterazine-hemission as a fraction of the total emission recorded during discharge inEGTA buffers covering the range of 0 to 40 µM free [Ca2+] containingescribed in Materials and Methods. Estimation of [Ca2+]i in vivo used

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Fig. 2.Developmental regulation of folate- and cAMP-induced[Ca2+]i responses. Dictyosteliumcells expressing recombinantapoaequorin were shaken in HL5 growth medium (1×106 cells/ml)containing 5 µM coelenterazine-h for 6 hours. To inducedevelopment cells were washed twice and shaken in MES-DB buffer(2×106 cells/ml) containing 5 µM coelenterazine-h. At the timesindicated, 1×108 cells were harvested, washed, resuspended in 5 mlbuffer containing 10 mM MES/NaOH, 10 mM KCl, 250 µM CaCl2,pH 6.2 and assayed for chemoattractant-induced [Ca2+]i changes inresponse to 10 µM folate (d) and 10 µM cAMP (m), respectively.

Table 1. Properties of folate- and cAMP-induced Ca2+

responsescAMP responses

Folate responses (aggregation-(vegetative cells) competent cells)

[Ca2+]i resting level (nM) 86±13 42±7(n=30) (n=28)

Peak [Ca2+]i in response to 172±22 203±43saturating stimulus (nM) (n=12) (n=12)

Time of onset (s) 8.3±1.0 9.2±1.2(n=30) (n=28)

Time of maximum (s) 23.3±2.2 27.6±2.8(n=30) (n=28)

Measurements of intracellular free calcium levels in resting andchemoattractant-stimulated cells were carried out as described in Materialsand Methods. The time of onset and maxima for folate- and cAMP-inducedincreases were calculated from digitized data captured at 50 Hz. Mean valuesare given ± standard deviations.

pension an economical compromise was, therefore, addition of 2.5-5 µM coelenterazine (final concentration) to thincubation medium from a 5 mM methanolic stock solutiocontaining 20% (w/v) Pluronic F-127. Fig. 1A shows the timcourse of aequorin reconstitution in Dictyosteliumcells undersuch conditions. Reconstitution was completed after a ~6 hincubation, but sufficient amounts for reliable in vivo mesurements can be obtained within shorter incubation times (60 minutes).

The calcium concentration-effect curve of native aequois non-linear on a log-log plot and imposes a lower limit othe range of [Ca2+] detectable with aequorin as the Ca2+-inde-pendent light becomes a significant fraction of the total ligemission (Allen et al., 1976; Cobbold and Rink, 1987). Wconfirmed this for our recombinant aequorin reconstitutwith native coelenterazine in D. discoideum (Fig. 1B).Accordingly, native aequorin as an intracellular calciuindicator cannot be used to measure [Ca2+]i below 100 nM,and is imprecise up to about 300 nM. However, the develment of novel synthetic coelenterazine derivates wimproved light emitting properties has made it possible customize the photoprotein’s sensitivity for the measuremof very low [Ca2+]i (Shimomura, 1989; Knight et al., 1993)In order to test its usefulness we generated aequorin in vusing coelenterazine-h and determined a calibration curelating its rate of consumption over the relevant range of f[Ca2+]. It was found that this semi-synthetic aequorproduces an approximate parallel shift of the concentratieffect curve to the left by about 0.5 log units. Accordingly, tminimum detectable [Ca2+] is about 25 nM and the signal riseabout 200-fold if [Ca2+]i increases from 50 to 500 nM (Fig1B). Thus, the semi-synthetic aequorin generated by incubing Dictyosteliumcells in the presence of coelenterazineprovides a sensitive method for monitoring small chemotractant-induced changes in [Ca2+]i from low resting levels.

Developmental regulation of chemoattractant-induced responsesThe developmental regulation of folate- and cAMP-inducCa2+ responses is illustrated in Fig. 2. Maximal folatresponses were observed from cells that had just bharvested from growth medium and rapidly declined to lopersistent levels within 2 hours. The cAMP-induced Ca2+

response was undetectable in vegetative cells and graduincreased to detectable levels during the first 5 hours of svation. From 5 hours onwards the magnitude of the respostarted to increase dramatically, reaching peak levels betw8 and 10 hours and declined thereafter. The maximal cAresponsiveness of cells coincided with the formation of tigEDTA-stable aggregates, which indicated that they hacquired the aggregation-competent state.

Kinetics and magnitude of [Ca 2+]i responsesFig. 3A and C show the kinetics of folate- and cAMP-inducCa2+ responses in suspensions of vegetative and differentiaamoebae. The resting level of Ca2+i of vegetative cells wasfound to be significantly higher than that of aggregatiocompetent cells (Table 1). Addition of submicromolar concetrations of folate and cAMP to suspensions of vegetative aaggregation-competent cells, respectively, evoked a trans[Ca2+]i increase with temporal characteristics resembling tho

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of Ca2+ uptake measurements (Table 1). Typically [Ca2+]istarted to rise about 8 seconds after stimulation, rose rapidfor approx. 15 seconds to a peak and returned close to the pstimulus level within another 40-60 seconds. After largchemoattractant-induced responses [Ca2+]i sometimesremained at a new, elevated steady state level, while durichemotactic stimulation at concentrations below 50 nM wnoticed a retardation of the response. Frequently, we observan additional very brief [Ca2+]i increase that occurred inconcert with the injection of attractant. This was observed onin the presence of extracellular Ca2+, depended on the mechan-

2849Intracellular Ca2+ responses in Dictyostelium chemotaxis

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Fig. 3.Effect of folate and cAMPconcentration on the time course andmagnitude of chemoattractant-induced[Ca2+]i responses. (A,C) Temporal changesin [Ca2+]i in vegetative cells duringstimulation with folate (A) and inaggregation competent cells stimulatedwith cAMP (C). Signals were recordedfrom 1×108 cells suspended in 10 mMMES/NaOH, 10 mM KCl, 250 µM CaCl2,pH 6.2. The data show [Ca2+]i changes inresponse to addition of 6.4 nM (smallresponse), 160 nM (intermediate response)or 20 µM (large response) folate or cAMP.Arrows indicate the time point of additionof chemoattractant. (B,D) Dose responsecurves for chemoattractant-induced [Ca2+]iresponses. 5 ml suspensions(2×107cells/ml) of vegetative andaggregation competent cells werestimulated with folate (d) and cAMP (m)at the indicated concentrations. Data areexpressed as a percentage of the relativemagnitude of folate and cAMP-induced[Ca2+]i responses, respectively. Meanvalues and standard errors of 4independent experiments are presented.

Fig. 4.Real-time recordings of the average [Ca2+]i increase of a 5 mlsuspension (2×107cells/ml) of aggregation competent cells inresponse to serial cAMP stimuli of doubling magnitude. The externalbuffer contained 10 mM MES/NaOH, 10 mM KCl, 250 µM CaCl2,pH 6.2. The stepped line indicates the times of addition and theresulting changes in cAMP concentration (disregarding possibleeffects of cAMP and phosphodiesterase production by the cells).

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ical force of attractant injection rather than on the stimulus cocentration and also occurred in response to the injectionbuffer alone (results not shown).

The effects of folate and cAMP were dose dependent asaturated between 4 and 20 µM. Half-maximal activationoccurred at ~85 nM for folate and at ~125 nM for cAMP (Fi3B and D). The estimated average increase in [Ca2+]i evokedby a saturating stimulus of folate was about half as large asaverage maximum increase caused by cAMP (Table 1).

Adaptation of [Ca 2+]i responses to temporal cAMPstimuliRepeated addition of attractant at the same concentraelicited weaker or no further responses. This adaptationresponsiveness is one of the key features of chemota(reviewed by Devreotes and Zigmond, 1988). During serstepwise 2-fold increases in attractant concentration responses increased in magnitude to a maximum at cAconcentrations near the calculated KD for this attractant andprogressively declined at higher stimulus concentration up10−5 µM cAMP (Fig. 4). Nonetheless, over a >8,000-folrange in the absolute magnitude of the stimulus incremennM to ~40 µM), response magnitudes varied by only abo4-fold. This behaviour is analogous to chemotactic responin defined spatial gradients (Fisher et al., 1989), motilresponses to temporal gradients (Segall, 1988) and cArelay responses to incremental temporal gradients (Devreo1982). It indicates that the cells respond to relative rather th

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absolute changes of cAMP concentration, consistent with tfinding that the magnitude of the chemotactic respondepends on the increase in the fraction of occupied cAMreceptors.

2850

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B

Fig. 5.Dependence of chemotactic [Ca2+]iresponses on the extracellular Ca2+ concentration.(A) Aggregation-competent cells of the samebatch were stimulated with 10 µM cAMP. Prior toaddition of the chemoattractant cells were washedtwice and resuspended in MES-DB buffercontaining 250 µM Ca2+ (top trace), or innominally Ca2+-free MES-DB containing 2 mMBAPTA (bottom trace). (B) The relationshipbetween the magnitude of cAMP-induced [Ca2+]iincreases on [Ca2+] of the external medium. Allsolutions contained 10 mM MES/NaOH, 10 mMKCl, pH 6.9 in deionized water. [Ca2+] was set byadding Ca2+ from a 100 mM CaCl2 standardbuffer (Orion), or by adding 5 mM Ca-EGTAbuffer containing free Ca2+ in the range between10−4 and 10−5 M. Means of 2 independentexperiments are presented.

Inhibitors of Ca 2+-uptake and removal ofextracellular Ca 2+ abolish Ca 2+-responsescompletelyIncreases in [Ca2+]i arise in general from either Ca2+ entry viaCa2+-channels in the plasma membrane or via ligand-inducCa2+ release from intracellular stores. In order to investigawhich pathway accounts for the change in [Ca2+]i followingstimulation of Dictyosteliumamoebae with chemoattractants wanalysed the dependence of the change in [Ca2+]i on the presenceof extracellular [Ca2+] and its pharmacological profile. Loweringextracellular [Ca2+] to a level approaching ‘zero’ by replacingthe Ca2+-containing bathing medium with nominally Ca2+-freemedium containing 2 mM BAPTA (Fig. 5A) or 5 mM EGTA(not shown), completely inhibited the increase in [Ca2+]i. Thatthe observed Ca2+ responses depend on the extracellular [Ca2+]is further illustrated by the Ca2+-concentration-effect profile(Fig. 5B). In addition, we monitored [Ca2+]i responses aftertreatment of cells with Ca2+-channel blockers. Analysis of45Ca2+ uptake by resting and chemotactically stimulateamoebae identified a number of inhibitors of chemoattractainduced Ca2+ entry (Milne and Coukell, 1991). Here we usethree of the most effective of them, the organic Ca2+-channelblocker Ruthenium Red, the proton-specific ionophore CCCand the inorganic Ca2+-channel antagonist Gd3+. Fig. 6 showsthe effect of these inhibitors on cAMP-induced [Ca2+]i fluxes inaggregation competent Dictyosteliumcells. In agreement withprevious reports (Milne and Coukell, 1991), pretreatment w15µM Ruthenium Red, 15µM CCCP and 500µM Gd3+ causedcomplete inhibition of cAMP-mediated [Ca2+]i increases. Therewere no detectable changes in [Ca2+]i that could be related to aseparate release of the ion from intracellular Ca2+ stores.

DISCUSSION

In this article we focus on the dynamics of cytosolic free Ca2+

changes in chemotactically stimulated Dictyostelium cells,with the aim of investigating the potential role played by thsecond messenger in the signal transduction pathways reging amoeboid cell movement, chemotaxis and aggregatDirect demonstration of signal-response coupling via Ca2+

requires the measurement of temporal and local change

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[Ca2+]i during these processes in living cells. In recent yeathis has become possible with the development of a rangefluorescent Ca2+ indicators (Tsien, 1989) and recombinanCa2+-sensitive proteins (Prasher et al., 1985). However, theapplication in Dictyosteliumcells has met with limited successfor various technical reasons associated with the use of Ca2+-specific fluorescent dyes or with the limited sensitivity onative aequorin. Consequently, many reports investigating tputative second messenger role of [Ca2+]i during chemotaxisare contradictory. What does seem clear from these studiethat the cytosolic free [Ca2+] in Dictyosteliumamoebae issustained at a very low level and that only surprisingly smaif any, global changes in [Ca2+]i can be detected during chemo-tactic stimulation. In this work we adjusted the range oaequorin sensitivity to very low Ca2+ concentrations, allowingus to accurately monitor small changes at very low [Ca2+]i.

In the present study we estimated the resting level of [Ca2+]ito be ~85 nM in vegetative cells and ~40 nM in aggregativcells, in basic agreement with fura-2 measurements showthat the basal [Ca2+]i level in Dictyosteliumcells is about 50 nM(Schlatterer et al., 1992; Yumura et al., 1996). Upon addition nanomolar concentrations of folate or cAMP to stirred suspesions of vegetative or aggregation competent cells, respectivwe observed a rapid transient increase in [Ca2+]i. That theobserved folate- and cAMP-mediated [Ca2+]i responses are theresult of a receptor-activated process is shown by the fact tthe measured dose-response profiles, developmental regulapattern and adaptation properties correlate closely with treported characteristics of the respective chemoreceptors Wit et al., 1985, 1986; Hereld and Devreotes, 1992). Consistwith the hypothesis that folate and cAMP receptors couple wthe same Ca2+ uptake system (Milne and Coukell, 1991), bothfolate and cAMP-induced [Ca2+]i increases exhibited similartime courses, displayed identical dependence on the preseof extracellular Ca2+ and comparable sensitivities to inhibitionwith Ca2+ channel blockers.

The temporal characteristics of the reported chemoattractainduced [Ca2+]i increases agree with the kinetics of Ca2+-fluxmeasurements in cell suspensions by either 45Ca2+ uptake (Wicket al., 1978; Milne and Coukell, 1991), or with Ca2+-sensitiveelectrodes (Bumann et al., 1984; Menz et al., 1991). The mstriking resemblance is that the onset of the [Ca2+]i increase

2851Intracellular Ca2+ responses in Dictyostelium chemotaxis

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Fig. 6.Effect of inhibitors of Ca2+-uptake onchemoattractant-induced [Ca2+]i changes.Vegetative, or aggregation competent cells ofstrain AX2 were stimulated with 10 µM folate orcAMP, respectively. Prior to the experiment cellswere washed and resuspended at 2×107cells/ml in5 ml buffer containing 10 mM MES/NaOH, 10mM KCl, 250 µM CaCl2, pH 6.2. Cells werestimulated directly (control), or following a 2minute preincubation in the presence of 500 µMGdCl3, 15 µM Ruthenium Red or 15 µM CCCP, asindicated. Arrows illustrate the time point ofaddition of chemoattractant.

occurs after a lag phase of about 5-10 seconds following sulation, even in the presence of saturating stimuli. This brinduction period preceding Ca2+ influx could be due to severalbiochemical processes, or a slow process such as protein pphorylation being required for the activation (Milne and Couke1991). The subsequent rapid increase in [Ca2+]i culminates in asharp peak after about 25 seconds and rapidly returned closthe prestimulus level within 60 seconds. In response to a srating stimulus of cAMP, for example, a maximum responresulted in a [Ca2+]i increase from about 40 to approximatel200 nM (Table 1). This measurement represents an estimatthe mean global increase of [Ca2+]i in the population of cellsstudied. Therefore, this value is likely to be an underestimatethe true [Ca2+]i in locally restricted areas, such as directly undeneath the plasma membrane of individual cells.

The very fast [Ca2+]i response times reported by others moitoring [Ca2+]i (Saran et al., 1994; Yumura et al., 1996) cleardiffer from the characteristic kinetics of chemoattractaninduced Ca2+ uptake. We also detected additional fast, transieCa2+ spikes preceding the slow Ca2+-influx, which could beconsistent with the IP3-induced Ca2+ release seen in permeabilized cells (Europe-Finner and Newell, 1986a; Flaadt et 1993). However, these responses were much faster (pwithin 1 second, complete within 5 seconds) than thoreported by Yumura et al. (1996) and significantly faster ththe reported IP3 responses in Dictyostelium(Van Haastert etal., 1989). They were only observed in the presence of excellular Ca2+ and depended on the mechanical force injection rather than on the presence of attractant. They therefore unlikely to represent attractant-mediated intracellurelease of Ca2+. Since the response could be detected in tpresence of inhibitors of Ca2+ influx they are most probablydue not to Ca2+ influx but to aequorin release from celldamaged, for example, by filopodial breakage. Alternativethey may represent responses to mechanical stimulation inform of a fast Ca2+ influx through channels different from thoseinvolved in chemoattractant responses.

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Chemoattractant-mediated [Ca2+]i increases were dependenon the presence of extracellular free Ca2+, ([Ca2+]e), indicatingthat the observed responses are mediated by Ca2+-influx fromthe external medium. Ca2+ influx was inhibited in a concen-tration-dependent manner over a very narrow range wdecreasing [Ca2+]e from 10−4 to 10−5 M. Interestingly, K+

efflux in cAMP-stimulated aggregative suspensions displacomparable response kinetics and dose-response profiles to[Ca2+]i increases reported here and is inhibited if [Ca2+]e israised over a similar narrow concentration range (Aeckerle aMalchow, 1989). It therefore appears that inhibition of K+

efflux may be one of the possible roles of the observtransient [Ca2+]i increases at or above 100µM extracellularfree Ca2+. In addition to a dependence of the response [Ca2+]e we have demonstrated that after addition of Ca2+

channel blockers known to effectively inhibit chemoattractaninduced Ca2+ uptake no detectable amounts of Ca2+ arereleased from intracellular stores.

It has been reported that after a cAMP stimulus phosphopase C is activated (Bominaar and Van Haastert, 1994), tcytosolic IP3 concentrations transiently increase (Europe-Finnand Newell, 1987; Van Haastert et al., 1989) and that IP3 causesrelease of Ca2+ from non-mitochondrial stores (Europe-Finneand Newell, 1986a; Flaadt et al., 1993). These results led toexpectation that there would be a rapid IP3-mediated intracellu-lar Ca2+ response to cAMP. The present study shows that no srapid increases in [Ca2+]i occur after cAMP stimulation and thatthe Ca2+ responses that do take place are mediated by C2+

influx. It remains possible that the Ca2+ influx is accompaniedby a Ca2+ induced intracellular Ca2+ release. Such a responsewould also be abolished by any treatment preventing Ca2+ influx.Consistent with this, Dictyosteliumphospholipase C is of the δtype, thus Ca2+-dependent and presumably inactive at the Ca2+

concentrations present in unstimulated amoebae (Cubitt aFirtel, 1992). Ca2+ influx might activate the enzyme and evokthe IP3-mediated release of Ca2+ from intracellular stores. Onthe other hand, there is no evidence for changes in IP3 concen-

2852

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T. Nebl and P. R. Fisher

tration at the time of Ca2+ influx (Europe-Finner and Newell,1987; Van Haastert et al., 1989). Furthermore, it has been shthat chemotaxis and aggregation are normal in Dictyosteliumamoebae carrying a disrupted gene encoding phospholipa(Drayer et al., 1994), rendering it conceivable that IP3-mediatedCa2+ release does not play a role in chemotaxis.

The possiblity that influx, not intracellular release, mediathe Ca2+ signals involved in chemotaxis makes good biologicsense since, of the various second messengers, Ca2+ is the mostsuited, by virtue of its short diffusion range in the cytoplas(Allbritton et al., 1992), to carry a locally restricted signal thcould form gradients within amoeboid cells. IP3-mediatedresponses would reflect the distribution of IP3 itself, be thereforemore likely to be global and less likely to carry spatial gradiinformation for chemotaxis. Ca2+ influx through specific plasmamembrane Ca2+ channels in mammalian cells has been shownresult in highly localized [Ca2+]i increases within ‘microdomains’just underneath the plasma membrane and is known to selectactivate localized cellular responses such as neurotransmrelease in nerve terminals (Llinas et al., 1992). Yumura et(1996) recently presented evidence for the existence of a spaorganized Ca2+ gradient in polarized migrating amoebae adetected locally restricted [Ca2+]i increases within the rear corteof individual Dictyostelium cells following local stimulationwith a cAMP-filled capillary. Such localized changes in [Ca2+]imay mediate the spatially restricted rearrangement of acand myosin-filaments within the cortical cytoskeleton, thereregulating cell polarity and oriented locomotion.

Consistent with the idea that Ca2+ might play a role in regu-lating the dynamics of the Dictyosteliumcytoskeleton duringchemotaxis, the increases in [Ca2+]i reported here correlate temporally with that of actin depolymerization (starting at 5-1seconds and ending at ~20 seconds) and myosin II associwith the cytoskeleton (sharp peak at 20-25 seconds astimulus) (McRobbie and Newell, 1984; Hall et al., 1988; Land Newell, 1988; Nachmias et al., 1989; Yumura, 1993, 199It has been proposed that IP3 and Ca2+ stimulate actin poly-merization (Europe-Finner and Newell, 1986b; Newell et a1990) and that pseudopod extension results from this becof a locally restricted increase in [Ca2+]i at the leading edge ofthe cell (Malchow et al., 1982; Condeelis et al., 1988). Content with this theory, Unterweger and Schlatterer (199reported a significant reduction in oriented pseudopod extenin cells loaded with BAPTA (but also see Van Duijn and VHaastert, 1992). However, Yumura et al. (1996) recendemonstrated that cAMP stimulation of an aggregatiocompetent amoeba with a micropipette results in a rear-to-f[Ca2+] gradient, with little or no detectable Ca2+ response in thepseudopodium that is extended towards the pipette. Tcontrary to expectation, localized Ca2+ increases after stimula-tion occur not at the leading edge, but in the rear of the cell.obvious inference is that the observed [Ca2+]i increases mayprimarily mediate restricted, local depolymerization of acfilaments in the posterior of a cell. In support of such a roleCa2+, in vitro data show that Ca2+ activates the F-actin-frag-menting protein severin (Brown et al., 1982; Eichinger et 1991) and inhibits various actin crosslinking and bundliproteins (Condeelis and Vahey, 1982; Fechheimer and Tay1984; Witke et al., 1991; Prassler et al., 1997).

The data presented here provide direct evidence for chemtractant-induced [Ca2+]i responses in support of the hypothes

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that Ca2+ is a possibly important second messenger during Dic-tyostelium chemotaxis. Our results suggest that influx, nrelease, mediates the Ca2+ signals during chemotactic cellmovement. This transient influx would probably be maximal the membrane-cytoplasm interface where it could exceed threshold for induction of Ca2+-regulated changes in cytoskel-etal elements within spatially restricted areas. The availaspatio-temporal data on [Ca2+]i distribution render it likely thatone of its predominant roles is activation of Ca2+-sensitive actinfragmenting proteins and loss of actin bundling in the cell’s rewhich would hinder the formation of stable protrusions anthereby protect this region from pseudopod formation.

This work was supported by grants from the Australian ReseaCouncil. T.N. was a recipient of an Australian Postgraduate ReseaAward.

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(Received 8 July 1997 - Accepted 22 September 1997)