CellularMicrobiology(2001)3(2),6373MicroreviewGlidingmotilityandcellinvasionbyApicomplexa:insightsfromthePlasmodiumsporozoiteRobertMe nardLaboratoiredeBiologieetGe ne tiqueduPaludisme,InstitutPasteur,2528,RueduDocteurRoux,75724ParisCedex15,France.SummaryApicomplexaconstituteoneof thelargest phylaofprotozoa.MostApicomplexa,includingthosepatho-genictohumans,areobligateintracellularparasites.Theirextracellularforms, whicharehighlypolarizedandelongatedcells, sharetwouniqueabilities:theyglide on solid substrates without changing theirshapeandreachanintracellularcompartment with-out activeparticipationfromthehost cell. Thereisnowampleultrastructural evidencethat thesepro-cesses result from the backward movement ofextracellular interactions alongthe anteroposterioraxisoftheparasite.RecentworkinseveralApicom-plexa, includinggeneticstudiesinthePlasmodiumsporozoite, hasprovidedmolecularsupport forthis`capping'model.Itappearsthatthesamemachinerydrivesbothglidingmotilityandhost cell invasion.The cytoplasmic motor, a transmembrane bridge andsurfaceligandsessential for cell invasionarecon-servedamongthemainapicomplexanpathogens.Theapicomplexanzoite:aglidinginvaderApicomplexaareunicellular eukaryotesfoundinawiderange of vertebrate and invertebrate hosts. All areparasitic, andsomeareof majormedical andveterinaryimportance.ThemostdeadlytohumansisPlasmodium,the aetiological agent of malaria, which kills almost 3millionpeopleeachyear. Other generathat arepatho-genictohumansincludeToxoplasma,whichcausesmildinfections in most individuals but can induce severemalformationsinadevelopingfetusorencephalitisinanimmunocompromised host, and Cryptosporidium andIsospora,whichcancauseseverediarrhoea.Othersaremajor animal pathogens, including Eimeria, which causesheavy losses in domestic animals, and Babesia andTheileria, which are as devastating in cattle as the malariaparasitesareinhumans.At one or more steps in their life cycle, mostApicomplexa invade host cells. To enter cells, theseparasites transform into specialized stages, usually called`zoites',thathaveacommonstructural organization(seeFig.1 and the structure of a Plasmodium sporozoite as anexample).Zoites arepolarizedandelongatedcells.Theirshape is maintained by a set of microtubules anchored toananterior polar ringandrunninglongitudinallyunder-neath the parasite pellicle, which is composed of theplasma membrane and two inner membranes. Theiranterior pole (apical complex) contains secretory vesiclesknownasrhoptries(largeandclubshaped) andmicro-nemes(small androdlike). Theseorganelles,whichareunifyingstructural featuresof Apicomplexaandareonlyseenintheirinvasiveforms,secretetheircontentsattheanterior tip of the zoite during attachment to, and invasionof, the target cell. Cell invasion usually places the zoite ina parasitophorous vacuole, which does not fuse withendosomesorlysosomes.Insidethevacuole,zoitescaneither multiplybymultiplefissionor canrapidlylyseitsmembrane to exit the cell and gain access to a newlocation in the host. For example, Plasmodiumsporo-zoites invade secretory cells of the mosquito salivaryglands to reach the salivary duct, but they invadehepatocytes of the mammalian host to replicate andtransformintothenextparasitestage.Most Apicomplexa zoites are also motile. Although theydonot haveciliaor flagellaandcannot swiminliquidmedium, they can glide on solid substrates, including hostcell surfaces.Glidingmotility,specifictoApicomplexa,isdefined by the absence of any obvious modification in theshapeof themovingcell. Thisisunlikecrawlingmotilityof many animal cell types and amoeboid protozoans,which requires continuous remodelling of pseudopodiaor lamellipodiainthedirectionof movement. Althoughgliding is a very rapid mode of locomotion, reachingspeeds up to 10mm/s, Apicomplexa probably do not haveto glide to reach the target cell/organ but are insteaddrivenpassivelyviadigestiveorcirculatoryroutes.OnceQ2001Blackwell ScienceLtdReceived1August, 2000; revised5September,2000; accepted13September, 2000. *For correspondence. E-mail rmenard@pasteur.fr;Tel.(133)140613260;Fax(133)145688045.attached to the target cell/organ, however, zoites must bemotiletogainaccesstoanintracellularcompartment.CellinvasionandglidingmotilitybyApicomplexa:cappingatthezoitesurfaceThefirst stepinunderstandingthemechanismsof cellinvasionandglidingmotilitycamewithobservationsthatzoitescanrelocatemoleculesthat bindtotheir surface.Dubremetz and Ferreira (1978) found that cationizedferritin binds the entire surface of Eimeria sporozoites andrapidlyaccumulatesat their posterior pole. King(1981)reportedthat small latexbeadsareactivelytranslocatedalongthesurfaceandaccumulateattheposteriorendofgregarines, themost primitivegroupof Apicomplexa, aswell as of EimeriaandPlasmodiumsporozoites (King,1988). Backward movement of the surface label or beadsoccurredataspeedsimilartothatofforwardlocomotionof thezoite, andbothprocesseswereinhibitedbytheactin filament-disrupting drug cytochalasin (DubremetzandFerreira, 1978; Russell andSinden, 1981). Stewartand Vanderberg (1991) subsequently showed that glidingofthePlasmodiumsporozoitewasassociatedwithback-ward redistribution of its main surface protein, thecircumsporozoite (CS) protein. CS was found to besecreted on the zoite surface at its anterior end,translocatedposteriorlybyacytochalasin-sensitivepro-cess (Stewart and Vanderberg, 1991) and released on thesubstrate from the posterior trailing end of glidingsporozoites (Stewart and Vanderberg, 1988). Other zoiteshavenow beenfound to releasetrailsof surfacematerialthat delineate their gliding pattern on the substrate(Entzerothet al., 1989; Arrowoodet al., 1991; Dobro-wolski andSibley,1996).Thefirstdetailedultrastructural studiesofcell invasionby apicomplexan zoites have focused on red cell invasionbyPlasmodiummerozoites(Aikawaet al., 1978; Milleret al., 1979). These studies revealed that merozoitesenter red cells using a moving junction. After initialcontact, an intimate junction forms between the zoiteanterior endandthecell surface, whichisdrawnback-wardasaringaroundthezoitethatgraduallypenetratesintoanascent vacuole. Themembraneof thisvacuole,partially derivedfromthehost cell plasmamembrane,shapestheenteringzoiteandsealsoffatitsposteriortipwhenentryiscomplete. Asimilar junctionmovingfromanterior to posterior pole, sometimes visible as aconstrictionaroundtheparasite, hasnowbeenreportedduring cell invasion by zoites of many apicomplexangenera(JensenandEdgar, 1978; Russell andSinden,1981; Dubremetz et al., 1985; Entzeroth, 1985; Chobotaret al., 1993;Pimentaet al., 1994; Morisaki etal., 1995).Evidencehasnowaccumulatedthatintimateattachmentof thezoiteapex tothehost cell is tightly coupledtomicronemedischarge(Entzerothetal.,1992;CarruthersandSibley,1997;Carruthersetal.,1999),andinvagina-tion of the parasitophorous vacuole correlates with rhoptrydischarge(Sam-Yellowe,1996;Suss-Tobyetal.,1996).Fig.1.Plasmodiumlifecycleandultrastructureofthesporozoitestage.A.ThePlasmodiumlifecycleinvolvestwohosts,mosquitoandvertebrate,andgeneratesthreeinvasivestages,sporozoite,merozoiteandookinete.Sporozoitesareformedbeneaththemosquitomidgutfromanoocyst,amultinucleatestageoftheparasite,arereleasedintothebodycavityfluidthatcarriesthemtothesalivaryglands(SG)andawaittransmissioninsidethesalivaryduct.Inthevertebratehost,theydevelopinsideskinmacrophagesinspeciesthatinfectavianhosts,andinsidehepatocytes(HEP)inspeciesthatinfectmammalianhosts.Merozoites,formedinsidehepatocytesandredbloodcells(RBCs),invadeRBCs.Ookinetes,formedfromzygotesinthemosquitomidgutlumen,traversethemidgutepitheliumandtransformintooocysts.B.Theanteriorpoleofthesporozoitecontainselectron-dense,roundorelongatedorganelles(arrows),i.e.micronemesandrhoptriesrespectively.C.Cross-sectionofasporozoiteshowingthemicrotubulestructure,composedof15/16unitsarrangedaroundthetwo-thirdsoftheperipheryand1intheremainingone-third(arrow),lyingunderthepellicle.D.Longitudinal sectionofasporozoiteshowingtheplasmamembrane,theinnermembranecomplexandasubpellicularmicrotubule.E.Theinnermembranecomplexconsistsofthetwoapposedmembranesofflattenedvesicles/cisternae.64 R.Me nardQ2001Blackwell ScienceLtd,CellularMicrobiology,3,6373These observations led Russell (1983) and King (1988)toproposeacappingmodel of glidingmotilityandcellinvasion in Apicomplexa. Parasite ligands would besecretedonthezoitesurfaceat theanterior pole, bindto the substrate or the host cell surface and betransported to the posterior pole. Binding to a fixedsubstratewouldleadtoforwardlocomotion, andbindingto cell-surface receptors would lead to penetration into thecell. Both processes were known to be sensitive tocytochalasins,implying thateitheractin polymerizationoractinfilaments arerequired(JensenandEdgar, 1976;RyningandRemington,1978;Milleretal.,1979;Russelland Sinden, 1981). By using cytochalasin-resistantmutantsinboththehost cell andToxoplasma, Dobro-wolski and Sibley (1996) have shown that actin dynamicsinthetachyzoite, but not thehost cell, arecritical forinternalization. This is likely to be the case for most zoites,which frequently take a few seconds to invade apparently`passive' cells.Thereis,however,atleastoneexceptiontothe cappingmodel of cell invasionin Apicomplexa.Theileriasporozoites, whicharepolarizedbut sphericalcellswithnoclearlydefinedapicalcomplex,donotglide.They invadetheir target (lymphoid) cells by circumfer-ential zipperingoftheparasiteandhostcell membranes(reviewedinShaw,1997).Onecappingmachinery,requiringthetransmembraneproteinTRAP,drivesglidingandinvasioninthePlasmodiumsporozoiteThePlasmodiumsporozoiteisthestageof themalariaparasiteinjectedbythemosquitovector intotheverte-brate host (Fig.1). Besides its major medical importance,anattractivefeatureof thePlasmodiumsporozoiteisitsunique capacity to invade multiple cells and glide throughvarious tissues in each of its hosts. In the mosquito,sporozoitesarecarriedtothesalivaryglandsandmustcross their basal lamina, the underlying secretory cell, thesecretory cavity and, finally, thesalivary duct (Sterlinget al., 1973; Pimenta et al., 1994). In the mammalian host,sporozoites are injected in the skin and their finaldestinationistheliver, wheretheydifferentiateintothenext stage within hepatocytes. The route to the liver,which is still unclear, probably includes additional cellinvasionevents.Sporozoitesmostprobablycrossvesselendothelia to reach the bloodstream in the skin (SidjanskiandVanderberg, 1997), andoncearrestedintheliversinusoidstheymaytraverseendothelial(Sinnis,1996)orKuppfer (Verhave et al., 1980) cells before reachinghepatocytes.Alternatively,theymayhijackmobileleuco-cytes(Vanderbergetal.,1990)andtravel tothelivervialymphatics (Vaughan et al., 1999; Krettli and Dantas,2000). What is clear is that Plasmodiumsporozoitesisolated frommosquito salivary glands are fit for thejourney: theyinvadevirtuallyanyadherent cell typeinvitroanddisplayvigorousglidingwhendepositedonanysubstrate,includinguncoatedslides.Onlytwosporozoitesurfaceproteinsareknown, CS(circumsporozoite protein; Nussenzweig and Nussenz-weig, 1985) and TRAP(thrombospondin-related anon-ymous protein;Robson et al., 1988; Rogers et al., 1992).CS, a glycosylphosphatidylinositol (GPI)-anchored pro-tein, is involved in sporozoite binding to the mosquitosalivary glands (Sidjanski et al., 1997) and to themammalian liver (Sinnis and Nussenzweig, 1996).TRAP is a transmembrane protein that carries in itsextracellular domain two well-characterized adhesivemodules, anAdomainof vonWillebrandfactor andatype1repeat of thrombospondin(TSR) (Fig.2). Usinggene-targeting technology in Plasmodium berghei, amalarial speciesthat infectsrodents, TRAPwasshowntohaveacrucial roleduringsporozoiteglidingandcellinvasion (Sultan et al., 1997). TRAP() parasites, createdat stages that replicate within rodent erythrocytes, have anormal lifecycleuntil sporozoitesarereleasedintothebody cavity of mosquitoes; sporozoites, however, areunable to invade mosquito salivary glands, to infect rodentliversafter intravenousinjection, toglideonslidesor toinvadeculturedcells.The direct role of TRAP in the capping activityassociated with these processes is nowsupported bylocalization and functionaldata. TRAPismainly storedinsporozoitemicronemes(Rogerset al., 1992) andaccu-mulatesasacapat thesporozoiteanterior poleuponcontact with the host cell (Gantt et al., 2000). Duringgliding motility, TRAP is detected on the sporozoitesurface as one or a fewbright rings (Fig.2) and isreleaseddiscontinuouslyonthesubstrateasatruncatedevoidofacytoplasmictail(Kappeetal.,1999).GeneticstudiesinP. berghei haveconfirmedthat surfaceTRAPhas a direct role in sporozoite gliding (Kappe et al., 1999).Deletionofthelast14residuesinthecytoplasmictail ofTRAPdoes notaffectits surfaceexposurebut drasticallymodifies the gliding pattern: whereas wild-type sporo-zoites glide in continuous circles, mutants display apendulummovementbyrepeatedlyglidingone-thirdofacircle and moving back to the original position (Fig.3). Asone-third of a circle is the distance expected to beachievedby cappingalongthelengthof thecrescent-shaped sporozoite, this phenotype suggests that themembrane-proximal partoftheTRAPtail issufficientfortip-to-tipredistributionof TRAP, whereasthedistal partensures its release at the posterior tip, probably viaproteincleavage.andisconservedinotherapicomplexanzoitesProteins structurally related to TRAP have been describedGlidingandcell invasioninApicomplexa 65Q2001Blackwell ScienceLtd,CellularMicrobiology,3,6373inthePlasmodiumookinete, thestagethat invadesthemosquitomidgut, aswell asotherapicomplexangenera(Fig.2). Like TRAP, they are transmembrane proteinsstored within micronemes. Their ectodomains containvarious combinations of A domain and TSR, exceptTRAPC1 of Cryptosporidiumthat lacks an A domain(Spano et al., 1998). Their cytoplasmic tails do not exhibitprimary sequence homology, but are of similar length(,45 residues), are acidic and possess a C-terminaltryptophan.Independent lines of evidence indicate that theseproteinshavesimilarfunctions.Toxoplasmagondii MIC2(TgMIC2) (Wan et al., 1997) is mobilized frommicro-nemes to theapical surfaceof the parasite uponintimateattachment to the host cell (Carruthers and Sibley, 1997),andco-localizeswiththemovingjunctionduringtachy-zoite penetration of the host cell (Carruthers et al., 1999).Replacement of thecytoplasmictail of TRAPbythat ofTgMIC2inP. berghei sporozoitesdoesnot affect theirgliding, invasive or infective capacities, demonstratingthat these two proteins are functional homologues (Kappeet al., 1999). Plasmodium CTRP, produced by theookinetestage,probablyplaysasimilarrole.Inactivationof CTRP in P. berghei(Dessens et al., 1999; Yuda et al.,Fig.2.TRAPandAdomain,TSRandTRAPfamiliesofproteins.TRAP schematics. TRAP is a ,600-residue-long protein composed of: (i) a signal sequence, (ii) a 10-amino-acid region conserved in all species ofPlasmodium that infect mammals, (iii) an A domain, (iv) a thrombospondin type I repeat (TSR), (v) a series of amino acid repeats rich in proline andasparagine,(vi)atransmembranedomainand(vii)a ,45-residue-longcytoplasmictail (CT).A domain family of proteins. The A domain is a ,200-residue-long module that assumes an a/b fold with a solvent accessible metal ion-dependentadhesionsite(MIDAS)onitsupperface.Inintegrinachains,fiveresidues[DxSxS(,65)T(,30)D,orMIDASmotif]co-ordinateanion(Mg21orMn21)andareeachessential forligandbindingandintegrinactivation;theyareconservedinanumberofAdomains(1).TSRfamilyofproteins.TheTSRisa ,55-residue-longmotifwhoseconsensussequenceisx4WSxWx2Cx3Cx5RxRxCx11Cx9Cx4C.TRAPfamilyofproteins.Foreachprotein,thenumberofcopiesofAdomainandTSRareshown.TRAPonPlasmodiumsporozoitesurface.ImmunofluorescenceonliveP.berghei sporozoitesusingTRAPanti-repeatantibodies.Leftpanel,immunofluorescence1phase.66 R.Me nardQ2001Blackwell ScienceLtd,CellularMicrobiology,3,63731999) and Plasmodiumfalciparum(Templeton et al.,2000) showed that CTRP is necessary for ookinetetransformationintooocyst, alifecyclestepthat requiresookinetecrossingofthemosquitomidgutepithelium.It isalsoclear fromtheinterchangeabilityof thecytoplasmictailsof TRAPandTgMIC2that somecomponentsof acortical motorsystemareconserved.Themechanismofposteriorreleaseof TRAPisalsolikelytobeconservedinother zoites. Indeed, modificationinTRAPof theC-terminaltryptophan,presentin thecytoplasmictailsofallrelated proteins described, is sufficient to transformsporozoite circular gliding into pendulum movement(Kappeet al., 1999). Accordingly, TRAPhomologuesinToxoplasma (TgMIC2) and Neospora (NcMIC2; Lovettet al., 2000) arealsoreleasedascytoplasmictail-lesstruncates. Theseresultsindicatethat atransmembraneprotein essential for both gliding and cell invasion, aswell asproductsinvolvedinitsdirectional redistributionand posterior release, are conserved in a number ofApicomplexa.Although direct evidence that TRAP(or any relatedprotein)bindstothecortical motorsystemislacking,thefindingsdescribedaboveindicatethat it might act asamotility receptor in a similar fashion to integrins in crawlingvertebratecells(Sheetzet al., 1998). Integrinsconnecttheextracellular matrixtotheactincytoskeleton. How-ever, theydiffusefreelyinthemembraneontheuppersurfaceof lamellawhentheyareunoccupied, andbindreversiblytotheretrograde-movingactinfilamentswhentheybindtoaligand(Felsenfeldetal., 1996). Whentheligandisacoatedbead,integrinsaredrawncentripetallyto a region adjacent to the cell nucleus. When the ligand isafixedsubstrate, integrinsremainstationaryasthecellmigratesoverthem(RegenandHorwitz,1992;Smilenovet al., 1999) andareleft onthesubstratebehindthemovingcell (RegenandHorwitz,1992).Integrinslinktheextracellular matrixtotheactinstressfibresat sitesoffocal adhesions, which form at the leading edge of the cellandserveaspointsof tractionduringcell translocation(Lauffenburger andHorwitz,1996).Itremainstobe seenwhether membrane sites enriched inTRAP(or homo-logue) serve as points of traction for gliding Apicomplexa.TheTSRandAdomainofTRAPactassporozoiteanchorstothehostcellsurfaceduringcellpenetrationRecent studies have shown that loss of function mutationsin each of the A domain and TSR of TRAP impairsporozoite invasion without affecting gliding motility(K. Matuschewski, A. C. Nunes, V. Nussenzweig andR. Me nard, submitted). Particularly, alteration of bothmodulesabolishessporozoiteinvasionintocellswithoutaffectingglidingmotility,showingthat thetwoprocessesinvolvedistinctextracellularassociations.ThetwoTRAPadhesivemodulesarerequiredfor cell invasionspecifi-cally, and most probably interact with cell-surfacereceptors.Remarkably,modificationsintheTRAPextra-cellular modules similarly affect sporozoite invasioninto mosquito salivary glands, rodent hepatocytes andFig.3.TRAP-dependentglidingphenotypes.Time-lapsemicrographsofP.berghei sporozoitesglidingonuncoatedglassslides.A.Thewild-typesporozoiteadoptsatypical `rigidarc' shapetoglideontheslide;locomotionessentiallyconsistsofglidinginrepeatedcircles.B.Replacementofthecytoplasmictail ofTRAPbythatofToxoplasmaMIC2doesnotaltertheglidingphenotypeorcell invasion.C.Alaninesubstitutionoftheterminal tryptophanordeletionofthelast14residuesofTRAPcytoplasmictail abolishesinvasionandtransformsglidingintoa`pendulum' phenotype:thesporozoitecoversone-thirdofacircleandgoesbacktothestartingposition,repeatedseveral times.Thebasisofthebackwardportionofthependulumphenotypeisunknown;itmaybeduetothemotorsystemreversingitscoursewhenitreachestheparasiteposteriortipandisnotcleaved/disengaged,ortotheparasitebeingpassivelypulledbackbyinteractionswiththesubstratethatarenotcleavedoffduringforwardlocomotion.D.Deletionofthelast37residuesoftheTRAPtail abolishesbothcell invasionandglidinglocomotion.All TRAPmutantsaretransportedonthesporozoitesurface.Numbersindicateseconds.Glidingandcell invasioninApicomplexa 67Q2001Blackwell ScienceLtd,CellularMicrobiology,3,6373culturedcells, raisingthepossibilitythat thesporozoitemay rely on the TRAP dual ligand system for invading anycell it mayencounter. AsAdomain(s) andTSR(s) arepresent in most TRAP-related proteins, the correspondingzoitesmayalsousethemasabasicinteractionsystemwithhost cell surfaces. Thefamily member inCrypto-sporidium, TRAPC1, lacks an Adomain. Interestingly,enterocyte invasion by Cryptosporidium has featuresdistinct fromcell invasionbyother Apicomplexa. It isaslower processthat placestheparasiteintheso-calledextracytoplasmic (intracellular but peripheral) vacuole,andwhichappearstoresultfromtheparasitepullingthecell plasma membrane rather than penetrating into the cell(WardandCevallos,1998;Forneyetal.,1999).TheTSRisanadhesivemodulethat bindswithhighaffinity to the glycosphingolipid sulphatide and certainsulphated glycoconjugates (Holt et al., 1990). The TSR oftheCSproteinbindsheparansulphateonthesurfaceofhepatocytes and cultured cells (Cerami et al., 1992;Frevert et al., 1993; Sinnis et al., 1994), and similarfindings have been reported for the TSR of TRAP (Mulleret al., 1993). InteractionsbetweentheTRAPTSRandcell-surface heparan sulphate may be sufficient forsporozoiteinvasionbecauseTRAPlackingafunctionalAdomainstillpermits sporozoiteinternalization,althoughlessefficiently. Inaddition, modificationsof theTSRinTRAP(K. Matuschewski, A. C. Nunes, V. Nussenzweigand R. Me nard, submitted) and genetic or chemicalremoval of heparansulphate/glycosaminoglycanchainsfrom host cell surfaces (Frevert et al., 1996) only partiallyreducesporozoiteinvasionrates, whichfurthersupportsthenotionof adirect interactionbetweenthetwoduringinvasion.TheAdomainof TRAPalsobindsheparansulphate(McCormick et al., 1999). However, because of the muchgreater importance of the TRAPAdomain than cell-surfaceheparansulphatefor sporozoiteinvasion, theAdomain most probably binds to additional cell-surfacereceptor(s). Numeroustypesof moleculesareknowntobind to A domains. For example, the A (or I) domain of aMin aMb2 (CR3 or Mac-1), a leukocyte integrin thatmediates stable adhesion to venular endotheliumandextravasation, binds to intercellular adhesion moleculeICAM-1, heparan sulphates, complement fragmentiC3b,coagulationfactorsfactorXandfibrinogenandthehookworm-derived neutrophil inhibitory factor (NIF) (Uedaetal.,1994;Kamataetal.,1995;ZhangandPlow,1997;McCormicket al., 1999). TheAdomainof a2inintegrina2b1 (VLA-2), exposed on many cell types, binds tocollagen and laminin (Kamata et al., 1994; Dickeson et al.,1998;Emsleyetal.,2000).TheAdomainofTRAPmaysimilarly be able to bind to various molecules on cellsurfaces.Alternatively,itmayrecognizeanevolutionarilyconservedcell-surfacereceptor. For example, syndecans,whicharethemajor transmembranecarriersof heparansulphatechains(Bernfieldet al., 1999), areubiquitousonadherent cellsinmammalsandarefoundoninsect cells(Springet al., 1994), wouldbeideal partners, offeringentryintoawiderangeofhostcells.What motor system powers Apicomplexa gliding andhostcellinvasion?Experiments with cytochalasins have established that theparasite actin cytoskeleton is required for both gliding andinvasioninApicomplexa.InToxoplasmatachyzoites,thereference system for biochemical and cell biology work inApicomplexa, the majority of actin is found as monomericactinwithstrikinglylittleamountsof filamentousF-actin(Dobrowolski et al., 1997a), including in extracts fromglidingparasites(Poupel andTardieux, 1999). Immuno-electron microscopy has shown that actin is locatedmainlyat thetachyzoiteapexandinthespacebetweentheplasmamembraneandtheinnermembranecomplex(IMC) (seeFig.4; Dobrowolski et al., 1997b). Asimilaractin distribution was reported in Plasmodium merozoites(Pinderet al., 1998). Microfilaments, however, remainedundetectable in apicomplexan zoites by conventionaltechniques until jasplakinolide, a cell-permeant drugthat stabilizespreassembledactin, wasused(ShawandTilney,1999).Microfilamentswerevisualizedattheapexofjasplakinolide-treatedtachyzoites,sometimesorganiz-inganapical projection. Importantly, jasplakinolidewasfound to block tachyzoite gliding and invasion (Poupel andTardieux, 1999), suggesting that not only de novopolymerizationbut alsorapiddeassociationof actinarerequiredfor theseprocesses. Accordingly, anumber ofactin-binding proteins that can inhibit actin assembly and/or destabilize actin filaments have been identified in theseorganisms, such as (i) a depolymerizing factor inToxoplasma homologous to the ubiquitous ADF/cofilinfamily (Allen et al., 1997), (ii) toxofilin, a novel actin-binding protein that sequesters actin monomers and capsfilament ends (Poupel et al., 2000) and (iii) a Hsp70/32/34molecularcomplexinPlasmodiummerozoitesthatshort-ensactinfilamentsinvitro(Tardieuxet al., 1998)andissimilar toacomplexinvolvedinorientedmovement inDictyostelium(Hugetal.,1995).Forglidingandinvadingcells, apicomplexanzoites maythus relyon aparticularlydynamic cortical actin network, which may generatelocalizedactinpolymerizationandshortfilaments.It isdifficult toconceive, however, that actinfilamentassemblyperseissufficient toprovidethedrivingforcefor zoitelocomotion. Instead, asfirst proposedbyKing(1988), actin filaments probably form a scaffold for myosinmotors. Several myosinshavebeenidentifiedinToxo-plasma(Tgmyo-Ato-D) andPlasmodium(Pfmyo-A) byPCRscreensusinggenericmyosinheadprimersor via68 R.Me nardQ2001Blackwell ScienceLtd,CellularMicrobiology,3,6373the sequence genome projects (Heintzelman andSchwartzman, 1997; Hettmannet al., 2000). They areunconventional myosins, whichcanserveduringnumer-ouscellularprocessesincludingcell movement(Mermalletal.,1998).However,noconventional,filament-formingmyosin (type II) has been identified. Apicomplexanunconventional myosinsarestructurallyuniqueandformanovel class(XIV)inthemyosinsuperfamily. Theyarethe shortest myosins described so far, lack the conservedIQmotifsthat typicallydefinetheneckregionandhaveshort andbasic tail domains.Immunolocalization studies,using antibodies directed against highly conservedmyosin head sequences, have revealed that myosinslocalize underneath the plasma membrane in a circumfer-ential pattern in both Toxoplasma tachyzoites (Dobro-wolski et al., 1997b) and Plasmodium merozoites (Pinderet al., 1998). ToxoplasmamyosinTgmyo-Aisdetectedasabright spot neartheapical poleinrestingparasitesand localizes intimately with the parasite cell plasmamembraneinapical patches inmovingparasites, con-sistent witha roleingliding motility (Heintzelman andSchwartzman, 1999). Tgmyo-A was recently shown to betargeted under the parasite plasma membrane viainteractionof itstaildomainwithamembrane-associatedsaturablereceptor (Hettmannet al., 2000). Themyosinroleisfurther suggestedbytheuseof myosinATPaseinhibitors, which block gliding and cell invasion byToxoplasmatachyzoites(Dobrowolski etal.,1997b).Althoughthereisevidencefor acortical actinomyosinmotorinApicomplexa, itsorganizationremainsspecula-tive. The motor must be located between the plasmamembraneandtheouter faceof theinner membranecomplex (IMC) (see Figs1 and 4), which is thought to playanimportant structural roleduringthecappingprocess.Freeze-fracture analysis has shown that the IMC ofapicomplexanzoitescontainslineararraysof`intramem-branousparticles' (IMPs)thatarearrangedlongitudinallyand follow the underlying microtubules. This arrangementnot only serves as a structural framework, by maintainingthe rigid shape of the zoite, but also provides directionalityof the capping process via the linear arrays of IMPs.Indeed,thehelical,screw-likepathof somezoitesduringglidingmatchestheir helicallycoiledmicrotubules/lineararrays of IMPs (D'Haeseet al., 1977; Dubremetz andTorpier, 1978; Russell and Sinden, 1981), suggesting thattheIMPsmayactasanchoragepointsforanaxial motorsystem. Dobrowolski et al. (1997b) havereportedthat inosmoticallyswollenToxoplasmatachyzoites,inwhichtheplasma membrane detaches fromthe IMC, myosin isdetected at the plasma membrane while actin remainsbound to the IMC. This would suggest a translocationmachinery in which myosins would bind the bridgingproteinandwalkalongadorsal trackof homopolaractinfilaments; when the bridging protein binds a fixedsubstrate, myosinactivitywouldthenresult infilaments(and zoite) translocation over the interaction. Alternatively,myosins may bind to the IMC and walk on ventral,stationary actin filaments attached to the bridging proteins.Insteadof aslidingmechanism, acontractionmechan-ismwouldnecessitatethepresenceof bipolar myosinsthat bridge two actin filaments, as in myosin II filaments. Incrawlingcells, twotractionmodelshavebeenproposedfor translocationof thecell bodyover extensionsof theleadingedge.Accordingtoatransportmodel,onesetofdorsal filaments is pulled forward by myosins walking overa stationary track of long, ventral filaments which arelinked to the substrate via bridging proteins. Such amechanismhas beendocumentedinpost-mitotic cellsspreadingover retractionfibres(Cramer andMitchison,1993, 1995; Cramer et al., 1997), aswell asfor growthcone locomotion (for a review, see Mitchison and Cramer,1996). Accordingtoamoretraditional actinmyosinIIcontractile mechanism, bipolar myosins pull on actinfilaments of opposite polarity; this generates corticaltensionthattranslatesintoforwardmovementofthecellwhen adhesive contacts with the substrate are stronger atthefrontofthecell.SuchascenariohasbeenproposedforcelllocomotioninDictyostelium(Jayetal.,1995)andin fish keratocytes (Svitkina et al., 1997). Interestingly, thelatter are rapidly moving cells that display persistentpolarization and nearly constant shape and velocity duringa gliding-like locomotion. Acontractile mechanismforapicomplexan zoite gliding would best fit the visibleconstriction of the parasiteat the tight junction duringhostcell invasion.Fig.4.Thepellicleofapicomplexanzoites.Thediagramshowstheorganizationofthezoitepellicleasdeducedbyfreeze-fractureanalysis.Thepellicleconsistsofaplasmamembrane(e)andthetwomembranes(m,i)oftheinnermembranecomplex(IMC),acontinuouslayerofflattenedvesiclesarrangedinlongitudinal rows.Threefractureplanesleadtothreepairsofcomplementaryfracturefaces(E,external face;P,protoplasmicface).Longitudinal arraysofintramembranousparticlesarefoundintheP-facesoftheIMC.Longitudinal linesofhigherparticledensityoverlaysubpellicularmicrotubules(t).Reproducedwithpermission.ThisdrawingistakenfromDubremetz,J.F.inTheProtozoanPhylumApicomplexa.Mehlhorn(ed.).SpringerVerlag.ISBN0387178384.Glidingandcell invasioninApicomplexa 69Q2001Blackwell ScienceLtd,CellularMicrobiology,3,6373ConclusionandperspectivesRecent work has provided molecular support for thetheory of an actomyosin-based capping mechanismdrivingglidingandinvasioninApicomplexa(King,1988),aswell asonenewpieceofthepuzzle.Weknowthatatransmembraneproteinessential for bothprocesses isconservedinanumberofapicomplexangeneraandthatitsadhesivemodulesmediateinternalizationintoawiderangeof host cells. Thecortical motor that redistributesthe protein along the parasite axis, however, is still poorlydefined, and the interactions involved in gliding loco-motionremainunknown.Asanessential bridgingproteintransits viamicronemes, theseorganelles arelikely tocontainother piecesof thepuzzle. Ahandful of micro-nemal proteins have been described in zoites wheremicronemes can be purified (reviewed in Tomley andSoldati, 2000). Most aresolubleproteinswithadhesivedomains such as degenerate TSR and EGF-like domains,which may play a role in initial host cellzoite adhesion oras additional components of the tight junction between thetwo cells. Some have been described as being transientlydispersedover thetarget cell surface; theymaypartici-pate in junction formation or may act on the cell tofacilitatezoitepenetration. Besidesthedissectionof themotor and extracellular interactions involved in gliding andhost cell invasion, questions arise as to how these eventsareregulated. Thefirst insightsintothesignallingpath-ways involved in microneme exocytosis have beenreported(CarruthersandSibley,1999),butthetrigger(s)remainunknown.Areglidingandinvasion,whichinvolvedifferent ligands, inducedby distinct signals andcouldthey engage different motors? How are micronemesecretion and the `capping' process finely tuned withrhoptry discharge and parasitophorous vacuole forma-tion? With the two accessible modelsof Toxoplasma andPlasmodium, whosegenomesarebeingsequencedandwhich offer a combination of biochemical and geneticapproaches, new insights should soon follow. It isreasonabletothinkthat Apicomplexauseuniquemole-cular machineries to move and penetrate cells, which maybe fundamentally different fromthose driving crawlingmotility. 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