Calcium-mediated protein secretion potentiates motility in Toxoplasma gondii

IntroductionToxoplasma gondii, the causative agent of toxoplasmosis, is amodel organism for the phylum Apicomplexa, which alsoincludes Plasmodium spp. (the causative agents of malaria) andCryptosporidium parvum (an agent of waterborne diarrhea).Members of this phylum are named for their specialized apicalend, which contains unique secretory organelles. Motility bythese obligate intracellular parasites is essential for the rapidinvasion of suitable host cells. Apicomplexans undergodirected motility despite a lack of locomotory organelles(King, 1988), and they invade host cells actively rather thanrelying on the host cell cytoskeleton (Dobrowolski and Sibley,1996). Genetic and biochemical studies indicate that thisunique form of locomotion, termed gliding motility, is drivenby coupling the translocation of surface adhesins to an actin-myosin motor beneath the parasite plasma membrane (Sultanet al., 1997). The velocity and direction of gliding motility havebeen shown to depend on actin polymerization (Wetzel et al.,2003), but the methods by which other aspects of motility areregulated are largely unknown.

Apicomplexans contain a conserved family of proteinscalled thrombospondin-related anonymous proteins (TRAPs)that serve as adhesins for parasite motility. TRAP familymembers are type 1 transmembrane proteins that contain anintegrin-like A domain and at least one thrombospondin type1 domain (Kappe et al., 1999). Deletion of TRAP inPlasmodium sporozoites demonstrates that the adhesinencoded by this gene is required for both gliding and invasion

(Sultan et al., 1997). The TRAP homologue expressed by T.gondii MIC2, is stored in secretory organelles termedmicronemes. Contact with the host cell causes fusion ofmicroneme vesicles with the plasma membrane, placing MIC2on the apical surface of the parasite (Carruthers et al., 1999b).During cell invasion, MIC2 is transported to the posterior ofthe parasite (Carruthers et al., 1999a), where it is cleavedwithin its transmembrane domain by protease(s) to release theextracellular domains (Brossier et al., 2003; Carruthers etal., 2000). Attempts to delete MIC2 in T. gondii have beenunsuccessful, suggesting that it is essential. Anothermicronemal protein, M2AP, aids the transport of MIC2throughout the secretory network (Rabenau et al., 2001).Parasites lacking M2AP (m2apKO) demonstrate an 80%reduction in rapid cell invasion, indicating that secretion ofMIC2 is required for efficient cell entry (Huynh et al., 2003).

Although the precise signals inducing microneme secretionare unclear, pharmacological studies indicate that parasitecalcium is involved (Carruthers and Sibley, 1999).Apicomplexans have multiple calcium stores, such asacidocalcisomes, the endoplasmic reticulum and themitochondria (Moreno and Docampo, 2003). Increasingintracellular calcium by treatment with alcohols (Carruthers etal., 1999b), calcium ionophores (Carruthers and Sibley, 1999)or ryanodine receptor agonists (caffeine and ryanodine) (Lovettet al., 2002), triggers microneme secretion in the absence ofhost cells. Chelation of intracellular calcium with BAPTA-AMblocks both microneme secretion and gliding motility (Lovett

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Apicomplexans such as Toxoplasma gondii actively invadehost cells using a unique parasite-dependent mechanismtermed gliding motility. Calcium-mediated proteinsecretion by the parasite has been implicated in thisprocess, but the precise role of calcium signaling in motilityremains unclear. Here we used calmidazolium as a tool tostimulate intracellular calcium fluxes and found that thisdrug led to enhanced motility by T. gondii. Treatment withcalmidazolium increased the duration of gliding andresulted in trails that were twice as long as those formedby control parasites. Calmidazolium also increasedmicroneme secretion by T. gondii, and studies with adeletion mutant of the accessory protein m2AP specifically

implicated that adhesin MIC2 was important for gliding.The effects of calmidazolium on gliding and secretion weredue to increased release of calcium from intracellular storesand calcium influx from the extracellular milieu. Inaddition, we demonstrate that calmidazolium-stimulatedincreases in intracellular calcium were highly dynamic, andthat rapid fluxes in calcium levels were associated withparasite motility. Our studies suggest that oscillations inintracellular calcium levels may regulate micronemesecretion and control gliding motility in T. gondii.

Key words: Calmidazolium, Adhesin, Gliding motility, Microneme,MIC2

Summary

Calcium-mediated protein secretion potentiatesmotility in Toxoplasma gondiiDawn M. Wetzel1, Lea Ann Chen1, Felix A. Ruiz2, Silvia N. J. Moreno2 and L. David Sibley1,*1Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA2Laboratory of Molecular Parasitology, Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign,2001 South Lincoln Avenue, Urbana, IL 61802, USA*Author for correspondence (e-mail: [email protected])

Accepted 17 August 2004Journal of Cell Science 117, 5739-5748 Published by The Company of Biologists 2004doi:10.1242/jcs.01495

Research Article

JCS ePress online publication date 26 October 2004

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and Sibley, 2003; Vieira and Moreno, 2000). However,chelation of extracellular calcium with BAPTA or EGTA hasno effect on either microneme secretion or parasite motility andcell invasion (Lovett and Sibley, 2003). In addition, host cellcalcium signaling is not required for parasite invasion (Lovettand Sibley, 2003). Studies using fluo-4 and video microscopydemonstrate that cytosolic calcium levels undergo dramaticchanges during parasite motility (Lovett and Sibley, 2003), butthe relationship between these oscillations and gliding is notunderstood.

Here we use calmidazolium (CAL) to explore the linkbetween calcium fluxes, MIC2 secretion and parasite gliding.Although CAL is marketed as a calmodulin inhibitor, it hasalso been shown to increase the levels of intracellular calciumin Dictyostelium discoidium (Schlatterer and Schaloske, 1996),Madin Darby canine kidney (MDCK) cells (Jan and Tseng,2000), smooth muscle cells (Sunagawa et al., 1998) andplatelets (Luckhoff et al., 1991). Our studies show that CALaffects oscillations in intracellular calcium that increase MIC2secretion and stimulate gliding motility in T. gondii.

Materials and MethodsChemicalsCaffeine and chlorpromazine were purchased from Sigma (St Louis,MO). BAPTA-AM [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, sodium salt], BAPTA and calmidazolium wereobtained from EMD Biosciences (San Diego, CA). Fluo-4 AM waspurchased from Molecular Probes (Eugene, OR). Trifluorazine andW7 were purchased from CalBiochem (EMD Biosciences, La Jolla,CA).

Parasite cultureT. gondii tachyzoites were maintained by two-day passage inmonolayers of human foreskin fibroblast (HFF) cells as describedpreviously (Morisaki et al., 1995). Wild-type RH strain, RH strainparasites expressing β-galactosidase (clone 2F) (Dobrowolski et al.,1997), mutants lacking M2AP (m2apKO) and the complementedclone (IC4) (Huynh et al., 2003) were used in some experiments.Parasites were isolated after host cell lysis, passed through a 3-µmfilter and washed with Hanks’ balanced salt solution (LifeTechnologies, Gaithersburg, MD) that also contained 0.001 M EGTA,0.01 M HEPES (HHE). All cultures were shown to be free ofmycoplasma with the GenProbe mycoplasma detection system(GenProbe, San Diego, CA).

Trail assayParasites were allowed to glide on FBS-coated coverslips and trailsdeposited on the substrate as described previously (Håkansson et al.,1999). To determine the effects of calcium on gliding, parasites weretreated with varying concentrations of calmidazolium, caffeine,chlorpromazine, W7, trifluorperazine (all from Calbiochem), BAPTA,BAPTA-AM or DMSO for 10 minutes prior to gliding. Treatedparasites were allowed to settle on coated coverslips and incubated at37°C for 15 minutes in the presence of agents. Cells were lightly fixedin 2.5% formalin-PBS and trails were visualized by staining with mAbDG52 conjugated to Alexa488 or Alexa594 (Molecular Probes,Eugene, OR). Average trail length in parasite body lengths(approximately 7 µm) was determined from five randomly selected63× fields that contained ~10 parasites per field from two coverslipsper experiment. Results were averaged from three separateexperiments (mean±s.e.m.). Wide-field fluorescence images werecollected with a Zeiss Axiocam and Zeiss Axiovision software v3.0,

then processed and merged with Adobe Photoshop v5.5 (AdobeSystems, Mountain View, CA).

Two-color invasion assayParasite invasion of HFF cells was quantified using a previouslydescribed two-color fluorescence assay that distinguishes extracellularfrom intracellular parasites (Carruthers et al., 1999a). In brief,following a 5-15 minute pulse for invasion, lightly fixed cells werestained with mAb DG52 to the parasite cell surface protein SAG1(conjugated to Alexa594) followed by detergent permeabilizationand re-staining with the same antibody conjugated to a secondfluorochrome (Oregon Green). The percentage of parasites that hadinvaded (green) was determined from five random fields of at least tenparasites per field for two coverslips per experiment. The results fromfour experiments were averaged (mean±s.e.m.).

VideomicroscopyGliding of parasites resuspended in Ringer’s solution was monitoredby time-lapse video microscopy as described previously (Håkanssonet al., 1999). Images were recorded with a Hamamatsu ORCA ERcamera (Hamamatsu Inc, Japan) using Openlab v3.0.9 (Improvision,Lexington, MA), cropped and saved as QuickTime movies (v5.0).Videos were used to calculate twirling speed, percentage of parasitesgliding per time period and duration of gliding. Percentages of glidingparasites and average duration of gliding were calculated using fiverandomly selected fields containing at least ten parasites from each oftwo coverslips per condition per experiment. The results from threeexperiments were averaged.

Parasites were loaded with 100 nM Fluo-4 AM (Molecular Probes)for 5 minutes at 37°C, centrifuged and resuspended in warm Ringer’ssolution. Parallel samples were treated with 1% FBS and 0.02%DMSO, 1 µM CAL or 1 µM caffeine, and video images were recordedover a 15 minute time period. Time-lapse phase and fluorescentimages were collected at two frames per second under low-lightillumination. Pixel intensity was analyzed with Openlabs 3.0.9. Theaverage duration (time between beginnings of successive cycles), peakto low (time between the highest signal and the lowest signal duringa cycle) and time between (time between successive peaks) wasanalyzed for each parasite as described (Lovett and Sibley, 2003). Anew cycle was defined if the pixel intensity of a given frame droppedbelow the lowest point of the previous cycle, and was followed by twoframes with pixel intensity above this lowest point. The intensity offluo-4 fluorescence was normalized by dividing the temporalfluorescence intensity (Ft) by the fluorescence intensity at the start ofeach cycle (Fo) as described previously (Torihashi et al., 2002).

SDS-polyacrylamide gel electrophoresis and western blottingSDS-PAGE was performed in 8% mini-gels under reducing conditionsand proteins were transferred to nitrocellulose membranes asdescribed previously (Carruthers et al., 1999b). Western blotting wasperformed using mouse anti-TgMIC2 monoclonal antibody 6D10(ascites, 1:10,000) (Carruthers and Sibley, 1999), rabbit anti-TgMIC4monoclonal antibody (1:5000) (Brecht et al., 2000), rabbit polyclonalanti-TgMIC5 antibody (1:5000) (Brydges et al., 2000) and/or rabbitpolyclonal anti-TgACT1 actin antibody (1:10,000) (Dobrowolski etal., 1997).

Microneme secretion assayParasites were treated with varying concentrations of inhibitors or 1%DMSO for 5 minutes. Secretion was stimulated by transfer to 37°Cfor 15 minutes followed by transfer to a wet ice bath. To determinethe effects of calcium on CAL-mediated secretion, samples weretreated with 100 µM BAPTA-AM or 1% DMSO for 5 minutes at 18°C

Journal of Cell Science 117 (24)

5741Calcium enhances Toxoplasma motility

before incubation at 37°C for 2 minutes. Alternatively, 100 µMBAPTA was added immediately before stimulation of secretion for 2minutes to minimize leaching of intracellular Ca2+ stores. Parasitesupernatants were separated from pellets by centrifugation at 14,000g at 4°C and run on SDS-PAGE gels along with dilutions of the cellpellet. Stimulation with 1% ethanol was used as a positive control forsecretion (Carruthers et al., 1999b). Accidental parasite lysis wasmonitored by the release of actin, which is 98% soluble in T. gondii(Wetzel et al., 2003). Secretion was quantified from western blotsusing a Fujifilm FLA5000 phosphorimager (Fujifilm Medical SystemsUSA Inc, Stamford, CT) and analyzed using Fujifilm Image Gauge4.0.

Measurement of parasite [Ca2+]iParasites were washed twice in buffer A (116 mM NaCl, 5.4 mM KCl,0.8 mM MgSO4, 5.5 mM D-glucose, and 50 mM HEPES at pH 7.4)and centrifuged at 500 g for 10 minutes at room temperature. Cellswere resuspended to a final density of 1×109 cells/ml in loading bufferwhich consisted of buffer A plus 1.5% sucrose and 6 µM fura-2/AM.The suspensions were incubated for 30 minutes in a 25°C water bathwith mild agitation. Subsequently, the cells were washed twice withbuffer A to remove extracellular dye. Cells were resuspended to a finaldensity of 1×109 cells/ml in buffer A and were kept on ice. Parasiteswere viable for several hours under these conditions. For fluorescencemeasurements, a 50 µl aliquot of the cell suspension was diluted in2.5 ml buffer A (2×107 cells/ml final density) in a cuvette and scannedin a Hitachi F-2000 spectrofluorometer. For fura-2 measurements,excitation was at 340 and 380 nm and emission was at 510 nm. Thefura-2 fluorescence response to intracellular calcium concentrationwas calibrated from the ratio of 340/380 nm fluorescence values aftersubtraction of the background fluorescence of the cells at 340 and 380nm as described (Grynkiewicz et al., 1985). [Ca2+]i was calculated bytitration with different concentrations of Ca2+-EGTA buffers (Morenoand Zhong, 1996). Concentrations of the ionic species and complexesat equilibrium were calculated by employing an iterative computerprogram as described (Moreno and Zhong, 1996). Traces shownare representative of three independent experiments conducted ondifferent cell preparations.

ResultsCAL treatment increases gliding and invasion byT. gondiiTo examine the effects of CAL on T. gondii motility, weanalyzed trail formation and cell entry after drug treatment.Parasites gliding on a substrate leave trails of surfacemembrane proteins and lipids that are readily visualized withimmunofluorescence microscopy (Håkansson et al., 1999).Migration across the substratum was increased by CAL in adose-dependent manner, as demonstrated by increased traildeposition by gliding parasites (Fig. 1A). The average lengthof trails formed after treatment with 10 µM CAL, a drugconcentration consistent with that used in other systems, wasdouble the length of trails of control parasites (Fig. 1B).Treatment with 10 µM CAL also led to a statisticallysignificant increase in parasite invasion into host cells (Fig.1C).

CAL increases the percentage of gliding parasites andthe duration of gliding As CAL stimulated motility and invasion in static assays, wenext examined parasites in real time to determine how this drug

potentiated gliding. Parasite motility can be divided into threebehaviors: twirling, circular gliding and helical gliding(Håkansson et al., 1999). T. gondii treated with 10 µM CALwere found to twirl (Table 1) and glide in a circular pattern(data not shown) at similar speeds to controls, which indicatedthat gliding velocity was not increased by CAL treatment.However, CAL treatment increased the length of time parasitesspent gliding by 78% as well as increasing the actual numberof parasites moving by 39% (Table 1).

CAL increases microneme secretion Previous studies have indicated that microneme secretion isessential for motility (Carruthers et al., 1999a). To determineif CAL directly stimulated microneme secretion by T. gondii,

Fig. 1. Effects of CAL on gliding and invasion by Toxoplasmagondii. (A) Indirect immunofluorescence microscopy demonstratingthat the average length of trails deposited during gliding increasedwith calmidazolium (CAL) treatment. Parasites were treated withDMSO (control) or 10 µM CAL and allowed to glide on serum-coated glass. Trails were visualized with anti-SAG1 mAb DG52conjugated to Alexa488. Bar, 5 µm. (B) Quantification of trailsdeposited in assay shown in A demonstrated that trail lengthincreased with 1 µM and 10 µM CAL treatment. Bars show averagetrail length in parasite body lengths (mean±s.e.m.). *A significantdifference (P�0.05) compared to control trail lengths, two-tailedStudent’s t-test. (C) Percentage of T. gondii invading host cellsincreased with CAL treatment. Parasites were treated with 1 µM or10 µM CAL or DMSO and allowed to invade HFF cells. A two-colorimmunofluorescence assay was used to distinguish betweenintracellular and extracellular parasites as described previously(Carruthers et al., 1999a). 1 µM cytochalasin D (CD) was used as anegative control for invasion. Bars show the average percentage ofintracellular parasites (mean±s.e.m.). *A significant difference(P<0.05) when compared to the percentage of control parasites thatinvaded host cells, two-tailed Student’s t-test.

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parasites were treated with varying concentrations of CAL.Cells were removed by centrifugation and the presence ofsecreted proteins MIC2 and MIC4 in supernatants wasexamined by western blotting. MIC2 is a convenient markerfor microneme secretion because the secreted 95-100 kDaform (sMIC2) is released into the supernatant, whereas theuncleaved 115 kDa form remains in intact cells (Carrutherset al., 2000). Micromolar concentrations of CAL stimulatedsecretion of both MIC2 and MIC4 when compared withparasites treated with DMSO alone as shown by western blot(Fig. 2A) and quantitative phosphorimager analysis (Fig. 2B).As actin is 98% globular in these parasites, its presence in thesupernatant was monitored to control for inadvertent celllysis. Cell lysis was typically 1% or less, as indicated bycomparison with dilutions of a parasite cell standard (%pellet).

Increased gliding due to CAL results from stimulation ofMIC2 secretion To confirm that the effects of calmidazolium on gliding weredue to its action on MIC2 and not on other adhesins we usedthe m2apKO strain, which has a specific defect in MIC2secretion (Huynh et al., 2003). CAL induced micronemesecretion by m2apKO at similar levels to RH strain parasites,as indicated by the release of the microneme protein MIC5(Fig. 3A,B). However, MIC2 secretion was not stimulated bymicromolar concentrations of CAL in the m2apKO strain (Fig.3A). Quantification of secreted MIC2 indicated that m2apKOparasites released tenfold less MIC2 than wild-type (RH strain)parasites treated with CAL (Fig. 3B). This result is consistentwith previous findings that in the absence of M2AP, secretionof MIC2 is specifically impaired (Huynh et al., 2003). Actinrelease due to accidental cell lysis did not significantly varybetween the wild type (RH) and m2apKO parasites and wasless than 5% (data not shown).

To examine the effects of CAL on motility by m2apKOparasites, we analyzed trail formation after drug treatment.Migration across the substratum by m2apKO parasites was notaltered by CAL and trail lengths remained similar regardlessof drug treatment (Fig. 3C). However, trail formation by IC4,the complemented clone of m2apKO, increased with CALtreatment to a similar degree as in the wild type (data notshown). Interestingly, m2apKO parasites were found to have aslight but statistically significant defect in trail formation.Collectively, these results indicate that CAL potentiatedgliding motility in wild-type parasites by increasing MIC2secretion.


Table 1. Effects of treatment with CAL on gliding by T. gondiiAverage time gliding continues

Average twirling velocity (360°/sec)* Average % parasites gliding (seconds)†

Control Exp 1: 0.477 27.33±2.62 11.86±1.44Exp 2: 0.567

10 µM CAL Exp 1: 0.476 37.67±3.15 20.18±0.59‡

Exp 2: 0.566Percent increase – 38.68% 77.43%

*Ten video recordings of motile parasites from two separate experiments were analyzed for control and CAL-treated parasites; values are means.†Ten video recordings of motile parasites were analyzed from each of three experiments for control and CAL-treated parasites; values are mean±s.e.m.‡Significantly different; P�0.05, two-tailed Student’s t-test.

Fig. 2. Effect of CAL on microneme secretion in T. gondii.(A) Western blot of parasite supernatants following stimulation ofsecretion at 37°C showed that secretion of MIC2 and MIC4 wereincreased by treatment with CAL. Stimulation with 1% ethanol wasused as a positive control for secretion. Supernatants were comparedto diluted lysates of cell standards (% pellets). Accidental parasitelysis was less than 1% as monitored by actin release. Identical blotswere probed with monoclonal antibody 6D10 (cellular MIC2 andsecreted MIC2; upper panel), rabbit polyclonal serum to MIC4(middle panel) and rabbit polyclonal serum to actin (lower panel).(B) Phosphorimager analysis of western blots demonstrated that theamount of MIC2 and MIC4 secretion increased with increasingconcentrations of CAL. Bars represent the average of fourexperiments (mean±s.e.m.). *A significant difference compared toDMSO levels, P<0.05; two-tailed Student’s t-test.


CAL increases intracellular calcium concentration [Ca2+]iin T. gondiiCAL is marketed as a calmodulin inhibitor. To determine if thismechanism was operating in T. gondii, we tested the effects ofseveral calmodulin inhibitors including chlorpromazine,trifluoroperazine and W7. Treatment with these agents did notincrease the length of trails made by gliding parasites (Table2), indicating that CAL probably affected motility by anothermechanism.

CAL is also known to increase intracellular calcium in avariety of cells. To determine whether CAL increased the[Ca2+]i in T. gondii, we used fura-2/AM to monitor calciumlevels in the parasite (Grynkiewicz et al., 1985). Increasingthe concentration of CAL stimulated progressively greaterincreases in [Ca2+]i when parasites were maintained in a buffercontaining 1 mM CaCl2 (Fig. 4A). In contrast, when parasiteswere incubated in a buffer containing 1 mM EGTA (to preventCa2+ entry), a modest rise in [Ca2+]i was observed at lowconcentrations of CAL (1-3 µM) whereas at higherconcentrations of CAL (>5 µM), [Ca2+]i decreased after a slightinitial rise (Fig. 4B). This result suggests that the drug madethe membrane permeable to calcium, resulting in calcium lossfrom the cell. However, CAL-treated cells were not labeled bypropidium iodide, indicating that the plasma membrane bilayerintegrity was preserved (data not shown). Although thisindicates that calcium efflux was not due to nonspecificmembrane permeabilization, it is possible that CAL inducedion exchange across the membrane, either by influencingchannels or pumps. To monitor the changes in [Ca2+]i directlyduring the influx of extracellular calcium, parasites wereinitially incubated in 1 mM EGTA (Fig. 4C). The slightincrease in [Ca2+]i observed after addition of CAL, wasgreatly augmented by further addition of 2 mM CaCl2 to theextracellular medium (Fig. 4C, trace b). Collectively, theseresults indicate that at low concentrations of CAL, [Ca2+]i wasincreased due to release of calcium from intracellular stores,whereas at higher drug concentrations calcium entered acrossthe plasma membrane from the extracellular medium.

The CAL-induced increase in motility and secretion isdue to an influx of extracellular calcium To determine the source of elevated calcium affecting secretion

Fig. 3. Effect of CAL on m2apKO T. gondii. (A) Western blotting ofmicroneme proteins released into supernatants indicated thatm2apKO parasites were unable to secrete MIC2 even after CALtreatment, unlike wild-type parasites (RH strain). Stimulation with1% ethanol was used as a positive control for secretion. Supernatantswere compared to diluted lysates of cell standards (% pellets).Identical blots were probed with monoclonal antibody 6D10 (cellularMIC2 and secreted MIC2; upper panel) and rabbit polyclonal serumto MIC5 (lower panel). (B) Phosphorimager analysis of western blotsdemonstrated that the amount of MIC2 secretion by the m2apKOstrain was less than 10% of wild-type (RH strain) secretion even afterCAL treatment. However, secretion of the microneme protein MIC5was normal. Bars represent the average of three experiments(mean±s.e.m.). (C) Quantification of SAG1-labeled trails depositedin gliding assays demonstrated that average trail length formed bythe m2apKO strain did not increase with CAL treatment, unlike traillength in wild-type parasites (RH). 1 µM cytochalasin D (CD) wasused as a negative control for gliding. Bars show average trail lengthin parasite body lengths (mean±s.e.m.). *A significant differencewhen compared to control trail lengths; P�0.05, paired Student’s t-test.

Table 2. Effects of calmodulin inhibitors on glidingmotility in T. gondii

Condition Average trail length*

DMSO 1% 2.13 ±0.12Cytochalasin 1 µM 0.59±0.05Calmidazolium 1 µM 3.03±0.52Calmidazolium 10 µM 4.15±0.33Chlorpromazine 10 µM 2.01±0.28Chlorpromazine 100 µM 2.09±0.10Trifluoroperazine 10 µM 2.08±0.51Trifluoroperazine 100 µM 1.46±0.07W7 10 µM 2.26±0.67W7 100 µM 1.51±0.15

*Gliding motility was tested in the presence of inhibitors versus DMSOcontrol as described in the Materials and Methods. Values represent the meanlength of trails in parasite body lengths. Results shown are from threeseparate experiments, mean±s.e.m.

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and motility in CAL-treated parasites, we incubated parasitesconcurrently with CAL and BAPTA to chelate extracellularcalcium, or BAPTA-AM to sequester intracellular calcium.First, we examined whether the presence of extracellularcalcium affected CAL-stimulated motility as monitored by trailformation. We found that trail length no longer increased with

high doses of CAL if parasites were treated simultaneouslywith BAPTA (Fig. 5A). However, BAPTA can cause leakageof calcium from intracellular stores if cells are exposed to thechelator for prolonged lengths of time (Lovett and Sibley,2003). Therefore, we used a rapid secretion assay to examinewhether extracellular calcium was partly responsible for theeffects of CAL. First, parasites were treated with DMSOor varying concentrations of CAL at temperatures that werenon-permissive for secretion. Next, BAPTA was added andparasites were immediately placed at 37°C for 2 minutes tostimulate secretion. Cells were removed by centrifugation andsecretion was evaluated by detection of MIC2 in the


Fig. 4. Effect of CAL on intracellular calcium levels. (A-C) Parasiteswere loaded with fura-2/AM as described in Materials and Methods,and suspended in buffer A containing 1 mM CaCl2 (A) or 1 mMEGTA (B,C). (A) Increasing the concentration of CAL (added whereindicated by the arrow) stimulated progressively greater increases inthe amount of intracellular calcium when parasites were in buffercontaining calcium. (B) CAL was added where indicated by thearrow at the concentrations shown to the right. Incubating parasitesin the buffer containing EGTA promoted increases in intracellularcalcium at low drug concentrations but decreased intracellularcalcium at higher concentrations of CAL. (C) 1 µM CAL (b) or 2.5µl DMSO (a) were added where indicated by the first arrow and 2mM CaCl2 was added where indicated by the second arrow. CAL-stimulated release of Ca2+ from intracellular stores led to Ca2+ entryfrom the extracellular medium. The increase in calcium detected withsolvent alone was also observed in its absence and reflects thepresence of some extracellular fura-2.

Fig. 5. Effect of BAPTA on CAL-stimulated gliding and secretion.(A) Quantification of the length of SAG1-labeled trails deposited bywild-type parasites. Simultaneous BAPTA and CAL treatment didnot stimulate increased trail length, unlike CAL treatment alone. 1µM CD was used as a negative control for gliding. Bars showaverage trail length in parasite body lengths (mean±s.e.m.). *Asignificant difference when compared to control trail lengths;P<0.05, two-tailed Student’s t test. (B) Western blotting of parasitesupernatant proteins showed that CAL-stimulated secretion of MIC2was blocked by BAPTA. Stimulation with 1% ethanol was used as apositive control for secretion. Supernatants were compared to dilutedlysates of cell standards (% pellets). Blots were probed withmonoclonal antibody 6D10 (cellular MIC2 (cMIC2) and secretedMIC2 (sMIC2). (C) Phosphorimager analysis of western blotsdemonstrated that the amount of MIC2 secretion no longer increasedafter BAPTA treatment, even with increasing concentrations of CAL.Bars represent the average of three experiments (mean±s.e.m.).(D) Caffeine treatment did not increase length of trails formed byparasites during a gliding assay. 1 µM CD was used as a negativecontrol for gliding. Bars show average trail length in parasite bodylengths (mean±s.e.m.).


supernatants by western blotting (Fig. 5B). Treatment ofparasites with BAPTA blocked CAL stimulation of secretion,demonstrating that the influx of extracellular calcium wasindeed responsible for the effects of CAL (Fig. 5C). BAPTA-AM treatment blocked microneme secretion and glidingregardless of CAL concentration (data not shown), reflectingthe known requirement for elevated intracellular calcium forthese events (Lovett and Sibley, 2003).

Increased motility results from more rapid intracellularcalcium fluxes To determine if other agents that increase intracellular calciumlevels and microneme secretion potentiated gliding by T.gondii, we examined parasites treated with caffeine. Caffeineis thought to cause microneme secretion by stimulating releaseof calcium from IP3 or ryanodine-like receptors in T. gondii(Lovett et al., 2002). We found that caffeine did not increasetrail length in gliding assays (Fig. 5D). Thus, not all reagentsthat increased intracellular calcium and microneme secretionstimulated gliding.

To explore why calcium-mediated protein secretionstimulated gliding in CAL-treated but not caffeine-treatedparasites, we used fluo-4 to visualize qualitative changes incalcium levels during gliding by T. gondii. Calcium fluxescorrelate with gliding motility and often precede initiation ofgliding (Lovett and Sibley, 2003). We found that gliding bycontrol and CAL-treated parasites was generally associatedwith brightly fluorescent cells that underwent cycles ofoscillating fluorescence that gradually decreased in intensity,as reported previously (Lovett and Sibley, 2003). Aftertreatment with 1 µM CAL, parasites fluoresced even morebrightly (Table 3) and the frequency of oscillations increased(Fig. 6B and Table 3). Plotting kinetic changes in fluo-4fluorescence suggested that CAL-treated parasites had quickerfluxes in intracellular calcium (Fig. 6B, Table 3). Conversely,parasites loaded with fluo-4 and treated with 1 µM caffeineremained constantly bright (Fig. 6A,B and Table 3) and ceasedto glide after ~10 minutes (data not shown). Collectively, thesedata suggested that calcium transients, and not simplyincreases in intracellular calcium levels, are required forefficient gliding by T. gondii.

As the kinetic changes in fluo-4 fluorescence indicated thatCAL-treated parasites had more rapid but less intense fluxes inintracellular calcium (Fig. 6B, Table 3), we wished to explorethis relationship further. Therefore, we normalized the datashown in Fig. 6B by dividing the fluorescence intensity at each

point by the intensity at the start of each cycle (Torihashi et al.,2002). Transformation of fluo-4 intensities demonstrated clearand synchronized oscillations in intracellular calcium duringgliding by both control and CAL-treated parasites (Fig. 6C).Furthermore, normalization shows that levels of intracellularcalcium in CAL-treated parasites oscillate at twice the frequencybut half the amplitude of calcium levels in untreated parasites(Fig. 6C). Calcium oscillations in caffeine-treated parasites werenot apparent even after normalization (data not shown).

DiscussionCalmidazolium provides a useful tool for exploring theconnection between calcium fluxes, microneme secretion andgliding motility in the Apicomplexa. We show that CALpotentiates gliding motility and cell invasion by increasing theduration of gliding by T. gondii. The effect of CAL on glidingspecifically depends upon stimulation of MIC2 secretion.Treatment with CAL increased intracellular calcium levelsboth through release of calcium from intracellular stores andentry of calcium from the extracellular milieu. Finally, fluo-4studies demonstrated that whereas rapid oscillations in [Ca2+]icaused by CAL stimulated gliding, constantly elevatedintracellular calcium in parasites treated with caffeine resultedin microneme secretion without altering gliding. Collectively,our results indicate that oscillations in intracellular calcium arenecessary for efficient gliding by T. gondii.

Motility and cell invasion by apicomplexans are dependenton a family of conserved protein adhesins typified by TRAP.Disruption of TRAP, an orthologue of MIC2 in Plasmodiumberghei, results in nonmotile sporozoites (Sultan et al., 1997).In addition, replacement of the penultimate tryptophan andfinal asparagine of TRAP with alanine and serine results inmutants that undergo non-productive ‘pendulum’ gliding, inwhich sporozoites briefly move forward and then return to theiroriginal position (Kappe et al., 1999). MIC2 is a TRAPhomologue expressed by T. gondii and deletion of its bindingpartner m2ap compromises MIC2 secretion and rapid cellinvasion (Huynh et al., 2003). However, its involvement ingliding motility in T. gondii has never been demonstrateddirectly. Here we demonstrate that m2ap mutants also have adefect in trail formation and fail to upregulate gliding inresponse to CAL, demonstrating that MIC2 is involved inpromoting efficient gliding by T. gondii.

The precise calcium-regulated trigger of micronemesecretion by T. gondii has not been identified. In neuraltransmission, the synaptic vesicle protein synaptotagmin acts

Table 3. Duration of calcium transients during gliding in control, CAL- and caffeine-treated parasitesCategory Duration (seconds)* Peak to low (seconds)† Time between (seconds)‡ Average intensity§

DMSO 17.64±2.18 15.15±3.11 19.63±3.09 846.89±120.491 µM CAL 8.23±0.53¶ 5.30±0.50 8.30±0.12¶ 1350.86±524.911 µM caffeine 31.16±5.37 27.45±6.10 33.77±17.56** 1736.56±361.10

For DMSO and CAL, values are mean±s.e.m., n=9 (three parasites analyzed in each of three separate experiments). For caffeine, values are mean±s.d., n=5. *Average time between beginnings of successive cycles.†Time from the highest to the lowest intensity during a cycle.‡Average time between successive intensity peaks.§Average pixel intensity during gliding.¶Significantly lower; P�0.05, two-tailed Student’s t-test.**No oscillation, time period indicates duration of gliding.

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as the calcium sensor for triggering vesicle release (Sudhof,2002). Although an analogous mechanism may operate inparasites, homologues of synaptotagmin have not yet beendescribed in the Apicomplexa, despite the fact that several

complete genomes are now available (Gardner et al., 2002).Evidence also exists that activated calmodulin can serve as acalcium sensor in specialized secretory cells (Coppola et al.,1999; Park et al., 1997; Quetglas et al., 2002). Future studiesdescribing the upstream and downstream signaling eventsinvolved in calcium-mediated protein secretion by T. gondii arerequired to better understand the regulation of this uniqueprocess.

In most eukaryotic cells, regulated exocytosis requiresmobilizing calcium from internal stores, often combined withinflux of calcium from the external medium (Burgoyne andClague, 2003). Our previous studies indicated that intracellularstores within the parasite are largely responsible for the rise inintracellular calcium that accompanies normal motility andinvasion (Lovett and Sibley, 2003). Here we show that CALcan augment this process by also inducing influx ofextracellular calcium across the membrane (particularly athigher drug concentrations). Because this influx ofextracellular calcium is not due to non-specific membranepermeabilization, our results suggest that CAL affects calciumchannels in the parasite plasma membrane. In most eukaryoticcells, calcium gains access across the plasma membranethrough receptor-operated, voltage-gated and/or store-operatedcalcium channels (Moreno and Docampo, 2003). In general,eukaryotic cells actively export calcium through Na+/Ca2+

exchangers and a plasma membrane Ca2+-ATPase (PMCA)(Moreno and Docampo, 2003). A PMCA has been localized toboth the plasma membrane and the acidocalciosome of T.gondii (Luo et al., 2001). Thus, CAL may mediate the entry ofextracellular calcium into T. gondii by disrupting the activityof this pump at the plasma membrane.

Previous studies demonstrated that increased intracellularcalcium stimulates microneme secretion by T. gondii(Carruthers et al., 1999a; Carruthers et al., 1999b; Carruthersand Sibley, 1999; Lovett et al., 2002); however, the agents usedin these studies lead to a refractoriness in further secretion andblock motility and invasion. A major advantage of CALtreatment is that it elevates calcium yet still allows motility andinvasion to be examined for an extended period. Our data usingother calmodulin inhibitors indicate that the effect of CAL inenhancing T. gondii motility is unlikely to be due to inhibitionof calmodulin. Monitoring intracellular calcium levels duringgliding revealed that more rapid fluxes in intracellular calciumlevels are associated with the increase in gliding duration seenafter CAL treatment of T. gondii. As these oscillations areeliminated by caffeine stimulation of secretion and as caffeine-treated parasites do not form extended trails in a gliding assay,calcium fluxes appear to be necessary for the continuation ofmotility. Periodic increases of calcium levels, such as thosestimulated by CAL, are likely to lead to repeated rounds ofmicroneme secretion and continuation of gliding. At present,we are not able to monitor secretion directly in live cells, andthus it is impossible to determine if each oscillation isaccompanied by a separate round of microneme secretion.Prolonged calcium increase, such as one caused by caffeine,results in the rapid release of microneme proteins, followed bya refractory period where gliding is inhibited. Consistent withthis hypothesis, pre-treatment of parasites with agonists thatstrongly stimulate microneme secretion decreases subsequentcell invasion (Carruthers et al., 1999a).

Rapid oscillations in intracellular calcium levels that were


Fig. 6. Real-time calcium measurements in motile control and CAL-treated T. gondii. (A) Time-lapse images of calcium flux in DMSO, 1µM CAL and 1 µM caffeine-treated parasites. Treated parasites werelabeled with fluo-4 and allowed to glide. Parasites were observed byfluorescence and phase-contrast microscopy and recorded at 0.5second intervals. Shown are selected merged images with the timeelapsed between frames indicated in seconds. Bar, 5 µm. (B) Graphof absolute frame-by-frame fluorescence pixel intensity of onerepresentative movie per condition showing fluo-4 fluorescenceintensity oscillations during DMSO, CAL or caffeine-treated gliding.*The point at which the caffeine-treated parasite stopped gliding.(C) Normalized intensities of fluo-4 oscillations in control and CALmovies shown in B. Calcium oscillations (numbered) in CAL-treatedparasites occurred at twice the frequency but half the amplitude ofcalcium oscillations in untreated parasites. Ft/Fo, temporalfluorescence intensity of fluo-4 divided by the fluorescence intensityat the start of each cycle.


observed during gliding indicate that intracellular calciumrelease and reuptake mechanisms are very active in T. gondii.Unfortunately, very little is known about calcium pools or howcalcium release is regulated in the Apicomplexa. In manyeukaryotes, calcium is stored mainly in the endoplasmicreticulum, and its release is mediated by IP3 or ryanodinechannels. T. gondii is sensitive to antagonists and agonists ofboth of these channels (Lovett et al., 2002), suggesting they arepresent and active in these parasites. SERCA-type ATPases arecalcium transporters that refill ER stores of calcium in othereukaryotes. T. gondii is sensitive to thapsigargin (Carruthersand Sibley, 1999; Moreno and Zhong, 1996), an inhibitorof these transporters (Thastrup et al., 1990; Thastrup et al.,1989), although SERCA-type ATPases have not yet beencharacterized in this organism. In addition, calcium ATPaseson the acidocalcisome, a unique calcium storage organelle inlower eukaryotes, could participate in intracellular calciumrelease and reuptake in T. gondii (Docampo and Moreno,2001).

Thus far, only one other drug, jasplakinolide (JAS), has beenshown to increase gliding motility in the Apicomplexa. JAScauses unregulated actin polymerization in T. gondii leading toincreased velocity and random directional changes that resultin non-productive locomotion (Wetzel et al., 2003). CALtreatment does not affect filamentous actin levels in the parasite(data not shown), but instead stimulates productive gliding byincreasing intracellular calcium and leading to micronemesecretion. Oscillations in intracellular calcium levels appear topromote prolonged microneme secretion and lengthen theduration of gliding by T. gondii. Thus, regulation of glidingmotility in these parasites occurs on two levels: actinpolymerization and microneme secretion, each of which directsdifferent aspects of motility in the Apicomplexa.

We thank the students in the Biology of Parasitism Course (MarineBiological Laboratory, Woods Hole, MA) who performed thepreliminary experiments that led to this study. We also thank NaomiMorrissette for helpful discussions, Vern Carruthers (Johns HopkinsUniversity) for antibodies to MIC2AP and the m2ap knockout andcomplemented strains and Julie Suetterlin for expert cell cultureassistance. Supported by NIH Grant AI34036 (L.D.S.), the BurroughsWellcome Fund (L.D.S.), NIH Institutional Training Grant AI017172-19 (D.M.W.), and the Medical Student Summer Research Program(L.A.C.) at Washington University School of Medicine.

ReferencesBrecht, S., Carruthers, V. B., Ferguson, D. J., Giddings, O. K., Wang, G.,

Jaekle, U., Harper, J. M., Sibley, L. D. and Soldati, D. (2000). TheToxoplasma micronemal protein MIC4 is an adhesin composed of sixconserved apple domains. J. Biol. Chem. 276, 4119-4127.

Brossier, F., Jewett, T. J., Lovett, J. L. and Sibley, L. D. (2003). C-terminalprocessing of the Toxoplasma protein MIC2 is essential for invasion intohost cells. J. Biol. Chem. 278, 6229-6234.

Brydges, S. D., Sherman, G. D., Nockemann, S., Loyens, A., Dåubener,W., Dubremetz, J. and Carruthers, V. B. (2000). Molecularcharacterization of TgMIC5, a proteolytically processed antigen secretedfrom the micronemes of Toxoplasma gondii. Mol. Biochem. Parasitol. 111,51-66.

Burgoyne, R. D. and Clague, M. J. (2003). Calcium and calmodulin inmembrane fusion. Biochim. Biophys. Acta 1641, 137-143.

Carruthers, V. B. and Sibley, L. D. (1999). Mobilization of intracellularcalcium stimulates microneme discharge in Toxoplasma gondii. Mol.Microbiol. 31, 421-428.

Carruthers, V. B., Giddings, O. K. and Sibley, L. D. (1999a). Secretion of

micronemal proteins is associated with Toxoplasma invasion of host cells.Cell. Microbiol. 1, 225-236.

Carruthers, V. B., Moreno, S. N. J. and Sibley, L. D. (1999b). Ethanol andacetaldehyde elevate intracellular [Ca2+] calcium and stimulate micronemedischarge in Toxoplasma gondii. Biochem. J. 342, 379-386.

Carruthers, V. B., Sherman, G. D. and Sibley, L. D. (2000). The Toxoplasmaadhesive protein MIC2 is proteolytically processed at multiple sites by twoparasite-derived proteases. J. Biol. Chem. 275, 14346-14353.

Coppola, T., Perret-Menoud, V., Luthi, S., Farnsworth, C. C., Glomset, J.A. and Regazzi, R. (1999). Disruption of rab3-calmodulin interaction, butnot other effector interactions, prevents rab3 inhibition of exocytosis. EMBOJ. 18, 5885-5891.

Dobrowolski, J. M. and Sibley, L. D. (1996). Toxoplasma invasion ofmammalian cells is powered by the actin cytoskeleton of the parasite. Cell84, 933-939.

Dobrowolski, J. M., Niesman, I. R. and Sibley, L. D. (1997). Actin inToxoplasma gondii is encoded by a single-copy gene, ACT1 and existsprimarily in a globular form. Cell Motil. Cyt. 37, 253-262.

Docampo, R. and Moreno, S. N. J. (2001). The acidocalciosome. Mol.Biochem. Parasitol. 114, 151-159.

Gardner, M. J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R.W., Carlton, J. M., Pain, A., Nelson, K. E., Bowman, S. et al. (2002).Genome sequence of the human malaria parasite Plasmodium falciparum.Nature 419, 498-511.

Grynkiewicz, G., Poenie, M. and Tsien, R. Y. (1985). A new generation ofCa2+ indicators with greatly improved fluorescence properties. J. Biol.Chem. 260, 3440-3450.

Håkansson, S., Morisaki, H., Heuser, J. E. and Sibley, L. D. (1999). Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals anovel, biphasic mechanism of cell locomotion. Mol. Biol. Cell 10, 3539-3547.

Huynh, M. H., Barenau, K. E., Harper, J. M., Beatty, W. L., Sibley, L. D.and Carruthers, V. B. (2003). Rapid invasion of host cells by Toxoplasmarequires secretion of the MIC2-M2AP adhesive protein complex. EMBO J.22, 2082-2090.

Jan, C. R. and Tseng, C. J. (2000). Calmidazolium-induced rises in cytosoliccalcium concentrations in Madin Darby canine kidney cells. Toxicol. Appl.Pharmacol. 162, 142-150.

Kappe, S., Bruderer, T., Gantt, S., Fujioka, H., Nussenzweig, V. andMénard, R. (1999). Conservation of a gliding motility and cell invasionmachinery in apicomplexan parasites. J. Cell Biol. 147, 937-943.

King, C. A. (1988). Cell motility of sporozoan protozoa. Parasitol. Today 11,315-318.

Lovett, J. L. and Sibley, L. D. (2003). Intracellular calcium stores inToxoplasma gondii govern invasion of host cells. J. Cell Sci. 116, 3009-3016.

Lovett, J. L., Marchesini, N., Moreno, S. N. and Sibley, L. D. (2002).Toxoplasma gondii microneme secretion involves intracellular Ca2+release from IP3 / ryanodine sensitive stores. J. Biol. Chem. 277, 25870-25876.

Luckhoff, A., Bohnert, M. and Busse, R. (1991). Effects of the calmodulinantagonists fendiline and calmidazolium on aggregation, secretion of ATP,and internal calcium in washed human platelets. Naunyn SchmiedebergsArch. Pharmacol. 343, 96-101.

Luo, S., Vieira, M., Graves, J., Zhong, L. and Moreno, S. N. (2001). Aplasma membrane-type Ca2+-ATPase co-localizes with a vacuolar H+-pyrophosphatase to acidocalcisomes of Toxoplasma gondii. EMBO J. 20,55-64.

Moreno, S. N. J. and Docampo, R. (2003). Calcium regulation in protozoanparasites. Curr. Opin. Microbiol. 6, 359-364.

Moreno, S. N. J. and Zhong, L. (1996). Acidocalcisomes in Toxoplasmagondii tachyzoites. Biochem. J. 313, 655-659.

Morisaki, J. H., Heuser, J. E. and Sibley, L. D. (1995). Invasion ofToxoplasma gondii occurs by active penetration of the host cell. J. Cell Sci.108, 2457-2464.

Park, J. B., Farnsworth, C. C. and Glomset, J. A. (1997). Ca2+/calmodulincauses rab3a to dissociate from synaptic membranes. J. Biol. Chem. 272,20857-20865.

Quetglas, S., Iborra, C., Saskawa, N., Haro, L. D., Kumakura, K., Sato,K., Leveque, C. and Seagar, M. (2002). Calmodulin and lipid binding tosynaptobrevin regulates calcium-dependent exocytosis. EMBO J. 21, 3970-3979.

Rabenau, K. E., Sohrabi, A., Tripathy, A., Reitter, C., Ajioka, J. W.,Tomley, F. M. and Carruthers, V. B. (2001). TgM2AP participates in

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Toxoplasma gondii invasion of host cells and is tightly associated with theadhesive protein TgMIC2. Mol. Microbiol. 41, 537-547.

Schlatterer, C. and Schaloske, R. (1996). Calmidazolium leads to an increasein the cytosolic Ca2+ concentration in Dictyostelium discoideum byinduction of Ca2+ release from intracellular stores and influx of extracellularCa2+. Biochem. J. 313, 661-667.

Sudhof, T. C. (2002). Synaptotagmins: why so many? J. Biol. Chem. 277,7629-7632.

Sultan, A. A., Thathy, V., Frevert, U., Robson, K. J. H., Crisanti, A.,Nussenzweig, V., Nussenzweig, R. S. and Menard, R. (1997). TRAP isnecessary for gliding motility and infectivity of Plasmodium sporozoites.Cell 90, 511-522.

Sunagawa, M., Yokoshiki, H., Seki, T. and Sperlakis, N. (1998).Intracellular application of calmidazolium increases Ca2+ current throughactivation of protein kinase A in cultured smooth muscle cells. J. Vasc. Res.35, 303-309.

Thastrup, O., Dawson, A. P., Scharff, O., Foder, B., Cullen, P. J., Drobak,

B. K., Bjerrum, P. J., Christensen, S. B. and Hanley, M. R. (1989).Thapsigargin, a novel molecular probe for studying calcium release andstorage. Agents Actions 27, 17-23.

Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R. and Dawson, A.P. (1990). Thapsigargin, a tumor promotor, discharges intracellular Ca2+

stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase.Proc. Natl. Acad. Sci. USA 87, 2466-2470.

Torihashi, S., Fujimoto, T., Trost, T. and Nakayama, S. (2002). Calciumoscillation linked to pacemaking of interstitial cells of Cajal – requirementof calcium influx and localization of TRP4 in caveolae. J. Biol. Chem. 277,19191-19197.

Vieira, M. C. F. and Moreno, S. N. J. (2000). Mobilization of intracellularcalcium upon attachment of Toxoplasma gondii tachyzoites to humanfibroblasts is required for invasion. Mol. Biochem. Parasitol. 106, 157-162.

Wetzel, D. M., Håkansson, S., Hu, K., Roos, D. S. and Sibley, L. D. (2003).Gliding motility by Apicomplexans is regulated by actin filament assembly.Mol. Biol. Cell 14, 396-406.


Calcium-mediated protein secretion potentiates motility in Toxoplasma gondii

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