J. Cell Sd. 63, 311-326 (1983) 31Printed in Great Britain © The Company of Biologists Limited 1983

CALCIUM REGULATION OF FLAGELLATION INNAEGLERIA GRUBERI

FREDERICK L. SCHUSTER AND ROBERT TWOMEYDepartment of Biology, Brooklyn College, Brooklyn, New York 11210, U.SA.

SUMMARY

The amoeba-to-flagellate transformation of Naegleria gruberi was studied, exploring the role ofcalcium in control of this pattern of morphogenesis. A direct and an indirect role for calcium arepostulated based on experimental results. Direct inhibition in the presence of calcium is caused byionophore A23187, substances with ionophore-like activity (amphotericin) and the hormone cal-citonin, which facilitate calcium uptake into the cytosol. The indirect role is difficult to assess, butis believed to be related to calcium regulatory protein and its control of cyclic nucleotide levels inthe cell, based on inhibition by trifluoperazine. Calcium flux was studied by addition of 45Ca2+ tocell cultures, and tracing its movement during the transformation period.

Ultrastructural localization of calcium was attempted in amoeboid and flagellate stages, as wellas in reverting flagellates. Deposits that might represent calcium were observed under the plasmamembrane and, in calcium-induced reversion, in electron-dense spheres seen in the cytoplasm andat sub-membrane locations, suggesting expulsion of excess calcium.

INTRODUCTION

In its life-cycle, Naegleria gruberi can transform from an amoeba into a flagellate,a process that includes de novo synthesis of the flagellar apparatus. The flagellate stageis transitory and reversion to the amoeboid state occurs within a relatively short periodof time, or can be induced by various treatments. One of these treatments includesaddition of specific ions to the suspension medium (Jeffery & Hawkins, 1976). Will-mer (1956, 1958), one of the first to appreciate the value of the Naegleria transforma-tion as a means of studying differentiation, concluded that loss of essential ions wasresponsible for initiating transformation. Flagellation was suppressed by addition ofpotassium, sodium and calcium to the suspension medium. Willmer (1961) alsosuggested that cation loss at the time of flagellation might cause a change in the plasmamembrane, which would lead to pumping back of ions into the cell. More recently,interest has focused on calcium and its role in this transformation. Perkins & Jahn(1970) have proposed that cell surface calcium concentration was a controlling factorin transformation, through the Gibbs-Donnan equilibrium. According to Fulton(1977), release of calcium from intracellular reservoirs favours the amoeboid state,while reversal of calcium flow back into the reservoirs triggers assembly of themicrotubular cytoskeleton and other events leading to flagellation.

In the present study, we have examined the relationship between calcium andNaegleria flagellation, by using a number of compounds that, directly or indirectly,influence calcium activity and/or concentration in the cell. We have also attemptedto study directly the movement of calcium across the plasma membrane in flagellating

312 F.L. Schuster and R. Twomey

amoebal populations, by using the radionuclide 45CaClz. Finally, by application ofultrastructural cytochemical techniques, we have tried to localize calcium reservoirsinNaegleria amoebae and flagellates or, by treatment with specific agents, in revertingflagellates.

MATERIALS AND METHODS

The following strains of N. gruberi were used in this study: EGB , S and 1518/1. Amoebae weregrown on peptone/yeast extract/glucose agar with living bacteria (Klebsiella pneumoniae) as a foodsource. For flagellation studies, Petri plates were inoculated with amoebae and bacteria, and wereincubated for 24 h at 28 °C.

Cultures were harvested by flooding the agar surface with chilled ZraM-Tris (Tris(hydroxy-methyl)aminomethane) buffer (pH7-l) and dislodging amoebae from the substrate with anL-shaped glass spreader rod. Cells were washed twice in chilled 2mM-Tris, resuspended in 2mM-Tris, counted on a Coulter Counter, and adjusted for cell density to give populations of 5 X 105 to106 cells/ml. Suspensions of amoebae in 20 ml samples were transferred to 125 ml Erlenmeyer flasksand placed in a water bath shaker at 28 °C.

ChemicalsChemical agents used in the study were generally added to the amoebal suspension at the time of

addition of cells to the flask(s). Appropriate concentrations to be used in the experiments weredetermined by trial testing of a range of concentrations on flagellating amoebae. In general, thehighest concentration of the agent not overtly toxic for the amoebae was selected for further experi-mentation. In experiments in which early contact with the agent was desired, the agent was addedto the 2mM-Tris wash fluid used in flushing amoebae from the growth plate surfaces. In otherexperiments, agents were added at various times after cells were in the Erlenmeyer flasks andflagellation was under way. Chemicals used included: ionophore A23187 (Calbiochem); am-photericin B (as Fungizone, E. R. Squibb) and amphotericin methyl ester (gift from E. R. Squibb);tetrodotoxin (Calbiochem); verapamil (gift from Knoll Pharmaceutical); sodium, dibutyryl, andfree acid forms of cyclic adenosine monophosphate (Sigma); cyclic guanosine monophosphate(Sigma); cholera toxin (Calbiochem); forskolin (Calbiochem); caffeine (Sigma); carbonyl cyanide/>-trifluoromethoxyphenyl hydrazone or FCCP (Sigma); Taxol (gift from the National CancerInstitute), trifluoperazine (gift from Smith Kline & French); antimycin A (Sigma); EGTA or(ethylenebis(oxyethylenenitrilo))tetraacetic acid (Eastman). Several hormones tested included cal-citonin, deoxycorticosterone, progesterone and vasopressin (all obtained from Sigma).

Radionuclide labelling experiments

Amoebal populations were harvested (as previously described) and suspended in 20 ml ofpeptone/yeast extract medium prepared with dilute saline (Page, 1967) minus calcium. Theradionuclide 45CaCl2 (New England Nuclear: sp. act. 34-97 mCi/mg) was added to give a concentra-tion of 1 ^Ci/ml. Cells remained in labelled medium for approx. 12h, at which time the mediumwas decanted carefully so as not to disturb the attached amoebae, and chilled 2mM-Tris buffer wasadded. After agitating the flask to release cells from the flask surface, the cell population was washedtwice in Tris and treated as before, 20 ml of amoebal suspension were placed in 125 ml Erlenmeyerflasks. Duplicate 50^1 samples were taken from flasks containing flagellating amoebae, at intervalsover 60min. Sampling was extended (2—3 h) in several experiments. Samples were transferredeither to scintillation vials, or to filter discs (Whatman Scintillation Pad, no. 3MM). The filter discswere air-dried, washed with 0-01 M - E G T A to remove unbound 45Ca2+, and dried by successivetreatments in absolute ethanol: ether (1: 1), and ether. Dried filter discs were inserted into scintilla-tion vials. Scintillation fluid was added to both fluid- and filter-containing vials, and vials countedin a Beckman scintillation counter (model LS-150).

Electron microscopyCells were fixed for electron microscopy by removing a sample of cell suspension from the flask,

Naegleria flagellation 313

and adding it to 2% glutaraldehyde in sodium cacodylate buffer, followed by secondary fixation incacodylate-buffered osmium tetroxide. Following dehydration with ethanol, cells were embeddedin Spurr resin. Special electron-microscopic cytochemical procedures used included pyroanti-monate staining for calcium localization (O'Brien & Baumrucker, 1980), and 1 % tannic acid addedto the fixative for visualization of ionophore-induced changes in flagellating cells (Shannon &Zellmer, 1981). Sections were examined in a Philips 300 electron microscope operating at 80kV.

RESULTS

Data reported in this section are averages of repetitive experiments performed, forthe most part, using the EGB strain of N. gruberi. Where differences appeared be-tween strains employed in experiments, such differences are noted. Ultrastructuraltechniques were applied only to the EGB strain of Naegleria.

Flagellation response

The basic response of an amoebal population to washing in 2 niM-Tris buffer isshown in Fig. 1. Though populations differed to some extent, flagellation percentagesof 90% and over were typical. The 5 strain peaked early (approx. 1 h) in terms ofnumbers of flagellates, while the maximum flagellation for EGB strain occurred atapprox. 2h. These basic curves from repeated flagellation experiments will serve asthe control for the other experimental curves to be described. In practice, eachexperiment had its own control population for comparative purposes. A summary of

1 2Time (h)

Fig. 1. Standard curve showing flagellation in 2 mM-Tris buffer of two N. gruberi strains,EGB (9) and 5 (O). The time of maximal flagellation differs for each strain, though thepercentage flagellation is similar.

314 F. L. Schuster and R. Twomey

the different chemical agents employed, the concentrations used, and the effect(s) ofeach on flagellation is given in Table 1.

Trifluoperazine

Addition of trifluoperazine (TFP), at a concentration of 10 /XM, at the time amoebae

Table 1. Effects of various substances tested on N. gruberi amoeba-to-flagellatetransformation

Substance Concentration Effect

±±

CalciumEGTA

Ionophore A23187+calcium+EGTA

Amphotericin B+calcium

Amphotericin methyl ester+calcium

TetrodotoxinVerapamil

10mM1 mM

Ionophores and quasi-ionophores

10mM1 mM

0-5^/ml10 mM5/ig/ml

10 mM

Ion blockers1/^g/mll^g/ml

Cyclic nucleotides and nucleotide stimulatorsCyclic AMP, free acid

+calciumDibutyryl cAMPSodium cAMPDibutyryl cGMPCholera toxin

+ NAD+calcium

Forskolin

Calcitonin+calcium

Deoxycorticosterone

Progesterone

Vasopressin

Antimycin ACaffeine

FCCPTaxolTrifluoperazine

1 mM10 mM

1 mM

1 mM1 mM

1 /^g/ml0-1 mM10mM

100 IM

Hormones2/ig/ml

10 mM1/fti

10/JM100 yM

1/JM

100 [MlOOOmU/ml

Miscellaneous agents10/M

1 mM10mM

1 IM10 fM10 ^M

±±

±±±

±

±

+ , Stimulatory; —, inhibitory; ± , neutral.

Naegleria flagellation 315

were suspended in the Erlenmeyer flask (to or start of flagellation experiment) wasinhibitory. The percentage of flagellates developing was reduced by about half. Asimilar experimental design for the 5 strain amoebae resulted in only slight inhibition;when TFP was incorporated into the Tris wash used to flush amoebae from the agargrowth plates (to — 45 min), inhibition of the S strain was similar to that of the EGBpopulation. Trifluoperazine did, however, appear to produce a delay in flagellationpeak for 5 amoebae (Fig. 2).

Cyclic nucleotides

Three forms of cAMP were tested for activity: dibutyryl, free acid and sodiumcAMPs, all at a concentration of 1 min (Fig. 3). The dibutyryl and sodium cAMPswere without affect on flagellation, as was cGMP. Treated populations behavedsimilarly to the controls. The free acid form of cAMP, however, was strongly inhibit-ory for the duration of the exposure. In a series of experiments in which calcium(10 mM) was added the inhibition was reversed and the flagellation response wasnormal. Added magnesium (10mM) was able to reverse inhibition, but not back tonormal (data not plotted). These reversal experiments were consistent with one lot offree acid cAMP but could not be duplicated with a second lot of nucleotide.

Cholera toxin/forskolinSince cAMP appeared to have an inhibitory effect on flagellation, substances with

100-1

1 2Time (h)

Fig. 2. Affect of 10 fiM trifluoperazine (TFP) addition on populations of flagellating Naeg-leria . EGB is inhibited by approx. 50 % upon addition of TFP at start of experiment ( • ) , but5 is virtually unaffected ( • ) . Addition of TFP to S cells at time of washing cells from agarsurface (t0 — 45 min) produces the same pattern of inhibition in 5 (O) as it does in EGB •

316 F. L. Schuster and R. Twomey

100-i

1 2Time (h)

Fig. 3. Addition of cyclic AMPs to transformingNaegleria. Dibutyryl (A) and sodiumcAMPs (O) have no affect upon flagellation, while free acid cAMP ( • ) is strongly inhibit-ory.

stimulatory effects on cAMP synthesis were tested. These included cholera toxin(l^g/ml) and forskolin (100/XM). Cholera toxin was inhibitory for EGB strainflagellation (Fig. 4). This inhibition, however, was reversible by addition of calcium(10 mM) along with the cholera toxin. Addition of nicotinamide adenine dinucleotide(NAD) which is split by cholera toxin (Holmgren, 1981), at a concentration of0-1 mM, appeared to block the toxin-induced inhibition.

Ionophore A23187

Addition of A23187 at a concentration of 5 (JM was without affect on flagellation,though higher concentrations were toxic to Naegleria. In the experiment presentedin Fig. 5, A23187 was added at 2h to an already flagellated Naegleria population.Ionophore plus calcium (lOmiw) promoted reversion to the amoeboid state, thoughcalcium (IOITIM) by itself had little effect on reversion. Combination of ionophore,calcium (IOITIM) and EGTA (1 mM) caused a dramatic reversion from the flagellatestate. Within a period of 5 min, the entire flagellate population became rounded, theflagella having been resorbed into the cytoplasm (see section on Ultrastructure,below). The cells remained in the rounded state until they began to disintegrate. If,instead of calcium (along with ionophore and EGTA), magnesium (10ITIM) wasadded, the same reversion occurred but the rounded cells soon became amoeboid andmoved about in a normal fashion.

Naegleria flagellation 317

100—1

8 0 -

co

CDn

2 0 -

1 2Time (h)

Fig. 4. Inhibition of Naegleria flagellation by cholera toxin at 1 fig/ml (A), and its partialreversal by addition of calcium ion ( • ) .

100 - |

Fig. 5. Effect of addition of ionophore A23187 at 2h to flagellating populations oiNaeg-leria. All populations are identical up to 2h. At that time, one flask receives calcium ( • ) ,a second calcium plus A23187 (A), and a third receives calcium plus A23187 and EGTA( • ) . The latter shows rapid reversion to the amoeboid state.

318 F. L. Schuster and R. Twomey

Radionuclide labelling

In an effort to trace the calcium flux during flagellation, cells were labelled with45Ca2+ prior to triggering the flagellation response. Data derived from these experi-ments are presented in Fig. 6 as the ratio of scintillation counts (in c.p.m.) of extra-cellular calcium/intracellular calcium (or Sx/Sz). Following the Tris wash and sus-pension of amoebae in flagellation buffer, the 45Ca2+ level in the medium decreasedrelative to internal 4SCa2+, to approximately 15min. At that time 4SCa2+ effluxoccurred (at approx. 25 min) followed by some degree of stabilization (at approx.30 min). Later samples (60 min and beyond) tended to exhibit an erratic pattern, withno consistent profile of calcium flux being observed. The period of calcium effluxcorresponds to the point at which a sharp increase in percentage flagellation took place(Fig. 6).

Miscellaneous agents tested

A number of other substances were tried, which are reported to have a direct orindirect effect on calcium level(s) in cells. Tetrodotoxin and verapamil (Rasmussen

4 -

r 8 0

3 -

2 -

- 6 0Oco

-40 2CDCD

- 2 0

I I I20 40

Time (h)

I60

Fig. 6. Study of 45Ca2+ flux in radionuclide-labelled, transforming cell populations. Theflux is plotted as a ratio of extracellular/intracellular counts (S1/S2) on the left ordinate( • ) . Flux was calculated by sampling of cells and supernatant fluid for scintillation count-ing. The right ordinate (O) traces percentage of flagellation during the time period of theexperiment.

Naegleria flagellation 319

& Goodman, 1977; Fleckenstein, 1974), which block calcium uptake by nerve cells(early and late blocks, respectively), were without affect with calcium added. Ampho-tericin B (as Fungizone) and the water-soluble amphotericin methyl ester were em-ployed as quasi-ionophores (Yoshioka & Inoue, 1981). By damaging the plasmamembrane of cells, these substances increase cellular permeability. When added alongwith calcium (10 mil), amphotericin compounds produced an effect similar to that ofionophore A23187.

Caffeine is reported to cause calcium ion release from intracellular vesicles thatsequester calcium (Kiehart, 1981). At 1 mM, no effect on flagellation was observed,but higher concentrations were inhibitory.

Antimycin A (IO^M) was inhibitory to flagellation. It has been reported to blockmitochondrial calcium uptake in cells (Borle, 1972). FCCP (IJUM), which acts torelease calcium from intracellular reservoirs (Chen, Babcock & Lardy, 1978), wasinhibitory for flagellation.

Taxol (lO^iM), a compound that has a stabilizing affect on microtubules (Schiff &Horwitz, 1980), was without affect on Naegleria, either in promoting the flagellatetransformation or maintaining the flagellate state once the transformation was com-pleted. In order to overcome any possible permeability delay in uptake of Taxol, thedrug was added to fluid cultures of amoebae 24 h prior to induction of the flagellationresponse. Thus, Taxol failed to stabilize the flagellate stage, and reversion to theamoeboid state occurred whether or not Taxol was present.

Pearson & Willmer (1963) tried a variety of hormones to test their affect on mem-brane transport and, indirectly, flagellation. A number of these hormonal substanceswere tested, along with several not employed by Pearson & Willmer (1963), but whichare reported to affect calcium ion changes in cells. Vasopressin or ADH (concn> 100munits/ml) had an inhibitory affect on flagellation; it is reported to increasecAMP in cells (Balaban&Mandel, 1979), and calcium efflux in rat hepatocytes (Chenet al. 1978). Calcitonin, which increases calcium uptake by cells (Borle, 1973), wasinhibitory only when calcium was present in the flagellation medium. Its actionappeared similar, therefore, to that of ionophore A23187 and the amphotericin com-pounds tested. Progesterone at low concentration (IJUM) stimulated flagellation; itsaffect was neutral at 10 /iM, but inhibitory at 100 paA. Progesterone is reported to lowercAMP level (Sadler & Mailer, 1980) and induce calcium release in Xenopus oocytes(Wasserman, Pinto, O'Connor & Smith, 1980). Deoxycorticosterone was stimulatoryat 1 [M, but inhibitory at higher concentrations (10 and 100 pBA).

Ultrastructure

Attempts made to localize calcium deposits in amoeboid and flagellate cells weregenerally disappointing. Evidence of calcium deposits was equivocal (see Fig. 7,which shows dense plaques under the plasma membrane). As already noted, applica-tion of A23187 plus calcium and EGTA resulted in flagellate reversion within 5 min.These cells were fixed and examined for ultrastructural changes that could beassociated with a combined calcium/A23187/EGTA effect. Evidence of resorbedaxonemal structures was seen, both at the surface of the reverting cells (as seen in Fig. 8)

F. L. Schuster and R. Twomey

Naegleria flagellation 321

and in the cytoplasm. Possible calcium reservoirs seen in the cytoplasm of these cellsare electron-dense spheres measuring between 100 and ISO nm in diameter. In favour-able sections, these spheres appear to contain a dense core separated from the enclos-ing wall by a space (Fig. 9). In reverting cells from the calcium/A23187/EGTA populations, these spheres (or related structures) are seen beneath the plasmamembrane (Fig. 10), as well as in positions outside plasma membrane, suggestingextrusion (Fig. 11).

DISCUSSION

The results of this study support the previous reports of Willmer (1956, 1958),Pearson & Willmer (1963) and Fulton (1977) that calcium has an active role in theamoeba-to-flagellate transformation in Naegleria. They also extend the data ofYuyama (1971), who examined the affects of agents that interfered with oxidativephosphorylation, and synthesis of protein and RNA. The data demonstrate activecalcium flux at the time of the transformation.

Addition of calcium ion by itself - even at the relatively high concentration of 10 ITIM

- had a modest effect in producing reversion to the amoeboid state (Jeffery & Haw-kins, 1976). By adding, along with the calcium, any of several different agents whoseeffect is to increase calcium uptake, reversion is enhanced. These substances includeionophore A23187, amphotericin compounds and the hormone calcitonin. Sincecalcium is known to cause dissociation of microtubules, it is reasonable to assume thatby elevating the cytoplasmic calcium level assembly of the flagellar apparatus wouldbe prevented or, if it had already formed it would be disassembled. Calcium level ispresumably controlled in these cells and it was to determine the pattern of control thatother chemical agents were tested for their affect(s) on flagellating populations ofamoebae.

The calcium regulatory protein, calmodulin, is now known to exercise control over

Fig. 7. Naegleria cell treated with pyroantimonate for calcium localization. Thetechnique produces electron-dense plaques beneath the plasma membrane. A mitochon-drion with interior vacuole is adjacent to the membrane. X53 000.

Fig. 8. Reverting flagellate from population treated with calcium plus ionophore A23187and EGTA. (The same type of experiment is plotted in Fig. 5, showing rapid reversionfrom the flagellate state.) The flagellar axoneme can be seen in oblique sections (arrow)but microtubular breakdown appears to be occurring. The electron-demise fuzz seen onareas of plasma membrane is a consequence of tannic acid-glutaraldehyde fixation (Shannon & Zellmer, 1981), employed to enhance A23187-induced extrusion of cell materials. X15 000.

Fig. 9. Section through reverting stage from calcium A23187-EGTA-treated populationof flagellates. Electron-dense spheres are seen in the cytoplasm, several of these showingsome internal structure. These dense spheres are thought to represent sequestered cal-cium. X45 000.

Fig. 10. Reverting flagellate from cell suspension treated as in Fig. 9. Dense spheres areseen below the plasma membrane, x50 000.

Fig. 11. Cell from population as in Figs 9 and 10, with electron-dense material extrudedfrom the cell. X50 000.

322 F. L. Schuster and R. Tvxmey

a number of cellular functions (Cheung, 1982; Klee, Crouch & Richman, 1980;Scharff, 1981), including microtubule assembly (Marcum, Dedman, Brinkley &Means, 1978). On the assumption that such a control pathway exists inNaegleria, thecalmodulin antagonist trifluoperazine (Weiss & Levin, 1978) was tested and found tobe inhibitory. Thus, it may be assumed that some manner of calcium regulation isoccurring in Naegleria at the time of flagellation and reversion to the amoeboid state.Fulton & Lai (1980) have detected two calcium-binding proteins, which aresynthesized during differentiation.

Activation of adenylate cyclase to produce cAMP is another role of calmodulin andthis, too, was investigated. The data obtained support a link between the effects ofcalcium that were observed and cAMP levels. Free acid cAMP blocked the trans-formation, though dibutyryl and sodium cAMPs were apparently neutral. The lattertwo forms of cAMP are reportedly more likely to penetrate the plasma membrane, andyet no effect was seen. Our results with free acid cAMP are similar to those reportedby Okubo & Inoki (1973), though these authors had difficulty in repeating their initialobservations (Okubo & Inoki, personal communication). We found that the free-acid-induced inhibition could be reversed by addition of calcium (IOITIM), but these datacould not be reproduced with a different lot of the free acid cAMP.

Since there appeared to be a linkage between cAMP level and flagellation, sub-stances known to promote cAMP production were tested. These included choleratoxin (Holmgren, 1981) and forskolin (Seamon & Daly, 1981). Consistent with apossible inhibition by elevated levels of cAMP, cholera toxin was inhibitory but theinhibition could be reversed by addition of calcium. Forskolin, however, was withouteffect. Caffeine, reported to inhibit phosphodiesterase activity (Kiehart, 1981) andthereby increase cAMP levels, was inhibitory at a concentration of 10 mM, though at1 mM it was without effect. Inhibition was shown by vasopressin (ADH), consistentwith its reported role in increasing cAMP (Balaban & Mandel, 1979) or stimulatingcalcium flux (Chen et al. 1978). The inhibitory effect of calcitonin (in the presenceof calcium ion) is presumably related to its role in increasing calcium uptake in cells(Borle, 1973). Progesterone, reported to lower cAMP levels through inhibition ofadenylate cyclase (Sadler & Mailer, 1980), gave varied results depending on theconcentration used. A low concentration (IJUM) was stimulatory, but a high con-centration (100/iM) was inhibitory. Deoxycorticosterone was stimulatory at a con-centration of 1 /iM, but inhibitory at >10|UM. These findings, based on affects ofprogesterone and deoxycorticosterone, confirm the data of Pearson & Willmer (1963),who found that low concentrations were stimulatory and high concentrations inhibit-ory.

Substances promoting calcium passage across the plasma membrane inhibitedflagellation. The calcium ionophore A23187 caused reversion to the amoeboid stagein these experiments, as also reported by Fulton (1977). A less specific type of calciumtransport across the membrane is produced by amphotericin, by creation of mem-brane channels in an otherwise intact membrane (Pressman, 1976). Naegleria spp.are particularly sensitive to this drug and its effects (Schuster & Rechthand, 1975).This quasi-ionophore activity (see also Yoshioka & Inoue, 1981) was found in the

Naegleria flagellation 323

present study, in which amphotericin B (and its less toxic water-soluble form, ampho-tericin methyl ester) were used at concentrations of 0-5 [Jg/m\ and 5 jUg/ml, respec-tively. Yoshioka & Inoue (1981) suggested that amphotericin B might cause calciumrelease from internal reservoirs in sea-urchin eggs, acting like ionophore A23187.Calcitonin, which by itself had no apparent affect on flagellation, was inhibitory in thepresence of calcium. Borle (1973) reported a possible role for calcitonin throughregulation of calcium uptake into the mitochondrial pool. In Naegleria, however,calcium efflux from reservoirs was not apparent in the presence of calcitonin, thoughthe hormone did appear to promote calcium uptake across the cell membrane, whencalcium was present in the flagellation medium.

Tetrodotoxin and verapamil were used because of their reported roles in blockingcalcium movement (Rasmussen & Goodman, 1977; Fleckenstein, 1974). These sub-stances were without apparent affect on Naegleria. Taxol, which has a stabilizingaffect upon microtubules (Schiff & Horwitz, 1980), was employed in an attempt toblock reversion to the amoeboid phase, thereby fixing Naegleria cells in the flagellatestate. In the presence of Taxol, flagellates reverted in parallel to those from control,non-Taxol-treated populations. In an effort to ensure Taxol penetration (and itsaffect), cells were maintained in Taxol-containing fluid medium for 24h prior towashing for flagellation induction. As before, the transforming and reverting phasesfollowed one another just as with the control populations. It is concluded, thus, thatTaxol is without affect in the Naegleria transformation/reversion.

Antimycin A is reported to block uptake of calcium by mitochondria, a processinvolving energy consumption (Borle, 1972). The compound inhibited flagellation,presumably by keeping cytosol calcium level high and preventing microtubule forma-tion. Carbonyl cyanide/>-trifluoromethoxyphenyl hydrazone (FCCP), by promotingcalcium release from its internal pool (Chen et al. 1978), also elevates the cytosoliccalcium level. FCCP, like antimycin A, was found to inhibit the flagellation response.

Though the various chemical agents employed appeared to bring about change inthe cytosolic calcium level, evidence for this change was indirect, revealing nothingof calcium flux at the time of flagellation. Thus, 45Ca2+ was used in order to examinethe flow of calcium accompanying the transformation. To this end, Naegleriaamoebae were labelled for approximately 12 h in 45Ca2+-containing medium to allowequilibration to take place (Borle, 1981). Cells were washed, both to eliminateunbound 45Ca2+ and to initiate flagellation. Calcium flux is presented in Fig. 6 as theratio of extracellular/intracellular counts per minute. The initial response of the cellpopulation was a relative increase in intracellular calcium, followed by calcium efflux.The point at which the shift over to efflux occurs coincides with the apparent shift inthe population from the amoeboid to the flagellate phase. The calcium efflux isconsistent with loss across the plasma membrane, either directly from calcium withinthe cytosol or indirectly from reservoirs containing the radionuclide. The consequentdrop in cytosolic calcium level would provide an ionic medium favourable tomicrotubule assembly and flagellation.

The evidence obtained from the experiments described suggests an active role forcalcium in flagellation and reversion. It was of interest to demonstrate the presence

324 F. L. Schuster and R. Tzuomey

of this cation supply in thin-sectioned material treated to exaggerate the calciumdeposits. In ultrastructural studies of amoeboid cellular slime mould stages by deChastellier & Ryter (1981, 1982), electron-dense deposits have been localized on thecytoplasmic face of the plasma membrane. Similarly, in the soil amoebaAcanthamoeba, electron-dense foci on the cell membrane are interpreted as sites ofcalcium sequestration (Sobota, Hrebenda & Przelecka, 1977; Sobota & Przelecka,1981a,b; Przelecka, Fritsch, Wollweber & Sobota, 1980). Using several of the stan-dard published techniques (e.g., pyroantimonate) for calcium localization, thepresence of such deposits in Naegleria was equivocal. With pyroantimonate, there issuggestion of calcium deposition under the membrane, in locations similar to thosein Dictyostelium and Acanthamoeba. In cell populations treated with ionophoreA23187, EGTA and calcium, there occurred a dramatic and rapid reversion to theamoeboid stage within 5 min, presumably involving an abrupt increase in cytosoliccalcium. Examining these cells in the electron microscope, one can easily see electron-dense deposits in the cytoplasm, generally as spheres with an internal substructure.Cells also show these dense bodies under the plasma membrane and outside themembrane. The latter location suggests expulsion of contents to the medium.

Our results support previous indications that calcium does indeed have a role in thetransformation of Naegleria. Our data suggest both direct and indirect involvementof calcium ion in morphogenesis. The direct role of calcium can be assumed from thedata on 45Ca2+ flux, demonstrating shifts in calcium ion across the membrane follow-ing flagellation induction. Substances known to affect calcium transport across themembrane, such as A23187 and calcitonin, substantiate a direct role for calcium.Amphotericin compounds, which non-specifically facilitate calcium flux across themembrane, also support a role for calcium.

The indirect involvement of calcium is more difficult to assess. However, on thebasis of a large body of literature implicating a calcium-regulatory protein in a varietyof cellular functions, we assume that this is also true in the case of Naegleria trans-formation. The inhibitory role of trifluoperazine supports such a relationship, as dothe results from the application of cyclic nucleotides and inhibitors or stimulators. Weare now in the process of combining studies of 45Ca2+ flux, with the testing ofionophores, trifluoperazine, etc., in an attempt to define better the direct and indirectroles of calcium.

The authors acknowledge the assistance of Mr Peter Feeney with portions of this research. ACUNY Research Award (13586) supported this work.

REFERENCES

BALABAN, R. S. & MANDEL, L. J. (1979). Comparison of the effects of increased intracellularcalcium and antidiuretic hormone on active sodium transport in frog skin. Biochim. biopkys. Ada555, 1-12.

BORLE, A. B. (1972). Kinetic analysis of calcium movements in cell culture. V. Intracellular calciumdistribution in kidney cells. J. Membrane Biol. 10, 45-66.

BORLE, A. B. (1973). Calcium metabolism at the cellular level. Fedn Proc. FednAm. Socs exp. Biol.32, 1944-1950.

Naegleria flagellation 325

BORLE, A. B. (1981). Pitfalls of the 45Ca uptake method. Cell Calcium 2, 187-196.CHEN, J-L. J., BABCOCK, D. F. & LARDY, H. A. (1978). Norepinephrine, vasopressin, glucagon,

and A23187 induce efflux of calcium from an exchangeable pool in isolated rat hepatocytes. Proc.natn. Acad. Sci. U.SA. 75, 2234-2238.

CHEUNG, W. Y. (1982). Calmodulin: An overview. Fedn Proc. Fedn Am. Socs exp. Biol. 41,22S3-22S7.

DE CHASTELLIER, C. & RYTER, A. (1981). Calcium-dependent deposits at the plasma membrane ofDictyostelium discoideum and their possible relation with contractile proteins. Biol. Cell 40,109-118.

DE CHASTELLIER, C. & RYTER, A. (1982). Calcium deposits at the plasma membrane of Dictyo-stelium discoideum during phagocytosis and cell motility. Biol. Cell 43, 121-128.

FLECKENSTEIN, A. (1974). Drug-induced changes in cardiac energy. Adv. Cardiol. 12, 183-197.FULTON, C. (1977). Intracellular regulation of cell shape and motility in Naegleria. First insights

and a working hypothesis. J. supramolec. Struct. 6, 13-43.FULTON, C. & L A I , E. (1980). Programmed appearance of translatable mRNAs for two calmodulin-

like proteins during cell differentiation in Naegleria. jf. Cell Biol. (abstr.) 87, 282a.HOLMGREN, J. (1981). Actions of cholera toxin and the prevention and treatment of cholera.

Nature, Land. 292, 413-417.JEFFERY, S. & HAWKINS, A. E. (1976). Studies of transformation in Naegleria gruberi: Effects of

ions and bacterial suspensions. Microbios 15, 27-36.KIEHART, D. P. (1981). Studies on the in vivo sensitivity of spindle microtubules to calcium ions

and evidence for a vesicular calcium-sequestering system. J . Cell Biol. 88, 604—617.KLEE, C. B., CROUCH, T. H. & RICHMAN, P. G. (1980). Calmodulin. A. Rev. Biochem. 49,

489-515.MARCUM, J. M., DEDMAN, J. R., BRINKLEY, B. R. & MEANS, A. R. (1978). Control of microtubule

assembly-disassembly by calcium-dependent regulator protein. Proc. natn. Acad. Sci. U.SA. 75,3771-3775.

O'BRIEN, T. L. & BAUMRUCRER, C. R. (1980). Ultrastructural localization of calcium in thelactating mammary cell by cytochemistry and X-ray microanalysis. Trans. Am. microsc. Soc. 99,403-415.

OKUBO, S. & INOKI, S. (1973). Control of flagellum formation in Naegleria gruberi. Bikenjf. 16,181-184.

PAGE, F. C. (1967). Taxonomic criteria for Umax amoebae, with descriptions of 3 new species ofHartmannella and 3 of Vahlkampfia.J. Protozool. 14, 499-521.

PEARSON, J. L. & WILLMER, E. N. (1963). Some observations on the actions of steroids on themetaplasia of the amoeba, Naegleria gruberi. jf. exp. Biol. 40, 493-515.

PERKINS, D. L. & JAHN, T. L. (1970). Amoeboflagellate transformations and the Gibbs-Donnanratio, j - Protozool. 17, 168-172.

PRESSMAN, B. C. (1976). Biological applications of ionophores. A. Rev. Biochem. 45, 501-530.PRZELECKA, A., FRITSCH, R. S., WOLLWEBER, L. & SOBOTA, A. (1980). Effect of a membrane-

stabilizing compound on calcium binding to the plasma membrane of Acanthamoeba castellanii.Acta histochem. 66, 181-191.

RASMUSSEN, H. & GOODMAN, D. B. P. (1977). Relationships between calcium and cyclic nucleo-tides in cell activation. Physiol. Rev. 57, 421-509.

SADLER, S. & MALLER, J. (1980). Progesterone prevents activation of adenylate cycles by choleratoxin \v\Xenopus oocytes.J. Cell Biol. (abstr.) 87, 169a.

SCHARFF, O. (1981). Calmodulin - and its role in cellular activation. Cell Calcium 1, 1-27.SCHIFF, P. B. & HORWITZ, S. B. (1980). Taxol stabilizes microtubules in mouse fibroblast cells.

Proc. natn. Acad. Sci. U.SA. 77, 1561-1565.SCHUSTER, F. L. & RECHTHAND, E. (1975). In vitro effects of amphotericin B on growth and

ultrastructure of the amoeboflagellates Naegleria gruberi and Naegleria fowleri. AntimicrobialAgents Chemother. 8, 591-605.

SEAMON, K. B. & DALY, J. W. (1981). Forskolin: A unique diterpene activator of cyclic AMP-generating systems. .7. cyclic Nucleotide Res. 7, 201-224.

SHANNON, W. A. JR & ZELLMER, D. M. (1981). Tannic acid-glutaraldehyde fixation revealscalcium ionophore-induced changes in rabbit polymorphonuclear leukocyte membranes. J.supramolec. Struct. Cell Biochem. 16, 155-165.

326 F. L. Schuster and R. Tzuomey

SOBOTA, A., HREBENDA, B. & PRZELECKA, A. (1977). Formation of calcium-dependent depositsat the plasma membrane of Acanthamoeba castellanii. Cytobiology 15, 259-268.

SOBOTA, A. & PRZELECKA, A. (1981a). Visualization of calcium-binding sites at plasma membraneof shock-frozen Acanthamoeba cells. Acta histochem. 68, 125-129.

SOBOTA, A. & PRZELECKA, A. (19816). Developmental changes in the localization of calciumbinding sites in Acanthamoeba castellanii. Histochemistry 71, 135-144.

WASSERMAN, W. J., PINTO, L. H., O'CONNOR, C. M. & SMITH, L. D. (1980). Progesterone

induces a rapid increase in Ca2+ in Xenopus laevis oocytes. Proc. natn. Acad. Sci. U.SA. 77,1534-1536.

WEISS, B. & LEVIN, R. M. (1978). Mechanism for selectively inhibiting the activation of cyclicnucleotide phosphodiesterase and adenylate cyclase by antipsychotic agents. Adv. cyclic Nucleo-tide Res. 9, 285-303.

WILLMER, E. N. (1956). Factors which influence the acquisition of flagella by the amoeba, Naeg-leriagruberi.J. exp. Biol. 33, 583-603.

WILLMER, E. N. (1958). Further observations on the "metaplasia" of an amoeba, Naegleriagruberi.J. Embryol. exp. Morph. 6, 187-214.

WILLMER, E. N. (1961). Amoeba-flagellate transformation. Expl Cell Res. (suppl.) 8, 32-46.YOSHIOKA, T. & INOUE, H. (1981). Activation of sea urchin eggs by a channel-forming ionophore

amphotericin B. Expl Cell Res. 132, 461-464.YUYAMA, S. (1971). The effects of chemical agents on the amoeba—flagellate transformation in

Naegleria gruberi. J. Prvtozool. 18, 337-343.

(Received 14 March 1983-Accepted 15 April 1983)