The 3D Structure of the Apical Complex and Association

14 Pages·2013·6.29 MB·English

The 3D Structure of the Apical Complex and Association
with the Flagellar Apparatus Revealed by Serial TEM
Tomography in Psammosapacifica, a Distant Relative of
the Apicomplexa
Noriko Okamoto*, Patrick J. Keeling
TheDepartmentofBotany,UniversityofBritishColumbia,Vancouver,BritishColumbia,Canada
Abstract
Theapicalcomplex is one ofthe defining featuresofapicomplexan parasites, including the malaria parasitePlasmodium,
where it mediates host penetration and invasion. The apical complex is also known in a few related lineages, including
severalnon-parasitic heterotrophs, whereit mediates feeding behaviour. Theorigin ofthe apical complex is unclear,and
onereasonforthisisthatinapicomplexansitexistsinonlypartofthelifecycle,andneversimultaneouslywithothermajor
cytoskeletal structures like flagella and basal bodies. Here, we used conventional TEM and serial TEM tomography to
reconstruct the three dimensional structure of the apical complex in Psammosa pacifica, a predatory relative of
apicomplexans and dinoflagellates that retains the archetype apical complex and the flagellar apparatus simultaneously.
The P. pacifica apical complex is associated with the gullet and consists of the pseudoconoid, micronemes, and electron
dense vesicles. The pseudoconoid is a convex sheet consisting of eight short microtubules, plus a band made up of
microtubulesthatoriginatefromtheflagellarapparatus.Theflagellarapparatusconsistsofthreemicrotubularroots.Oneof
the microtubular roots attached to the posterior basal body is connected to bypassing microtubular strands, which are
themselvesconnectedtotheextensionofthepseudoconoid.Thesecomplexconnectionswheretheapicalcomplexisan
extension of the flagellar apparatus, reflect the ancestral state of both, dating back to the common ancestor of
apicaomplexans and dinoflagellates.
Citation: Okamoto N, Keeling PJ (2014) The 3D Structure of the Apical Complex and Association with the Flagellar Apparatus Revealed by Serial TEM
TomographyinPsammosapacifica,aDistantRelativeoftheApicomplexa.PLoSONE9(1):e84653.doi:10.1371/journal.pone.0084653
Editor:StuartAlexanderRalph,UniversityofMelbourne,Australia
ReceivedJune29,2013;AcceptedNovember17,2013;PublishedJanuary2,2014
Copyright:(cid:1)2014Okamoto,Keeling.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbyagrantfromtheCanadianInsititutesforHealthResearch(MOP-42517)andfromtheTulaFoundationtotheCentrefor
MicrobialDiversityandEvolution.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction end;micronemes,whicharealsoelectrondensevesicles;anddense
granules,whicharesphericalvesicleslargerthanmicronemesand
The apicomplexans are a group of obligate intracellular
containing electron dense materials [9]. There is also diversity
parasitic protists that includes human parasites such as malaria
among the Apicomplexa, e.g., Plasmodium lacks the conoid or
parasites Plasmodium, a major disease for which about half the
Theilerialacksmicronemes.Theapicalcomplexisfundamental to
world population is at risk [1]. Other major human pathogens
apicomplexan infection, because itmediates the processes of host
include Toxoplasma gondii, which infects up to a third of world
attachment and invasion (for recent reviews see [10,11]).
population [2], and Cryptosporidium, a major contaminant of
Accordingly, the molecular components of the apical complex
drinking water. Apicomplexans also include important veterinary
and their role in the invasion machinery have been intensively
parasites, such as Eimeria (estimated to cause more than US $3
investigated based upon cell biological, genomic, transcriptomic,
billion annual loss worldwide to modern poultry industry [3]),
and proteomic information accumulated in the last decade
Cryptosporidium, Babesia, and Theileria, all of which cause economic
[12,13].
lossin varietyof livestock.
From an evolutionary perspective, the origin of the apical
Apicomplexan parasites are characterized by a subcellular
complex and its relation to other cytoskeletal components,
structure called the apical complex. The apical complex was first
especiallytheflagellarapparatusareofgreatinterest.Theflagellar
observed in thin sectioned Toxoplasma gondii under transmission
apparatusisafundamentalpartoftheeukaryoticcytoskeleton.Itis
electron microscopy (TEM) [4] and intensively studied in other
located at the base of the flagella and typically composed of the
apicomplexan parasites from the late 1950’s through the 1970’s
basalbody,microtubularroots,andfibrousconnectivestructures.
(forreview,[5–7]).TheapicalcomplexofT.gondiiiscomposedof
Moestrup [14] established a universal numbering system for the
the conoid, which is a closed, truncated cone composed of the
microtubular roots based on the generation of the basal bodies.
unique fibres of tubulin polymers [8] with terminal rings on the
This system enables us to use the flagellar apparatus to infer the
anterior apex; rhoptries, which are electron dense, rhomboid-
evolution of the system, and is accepted as a standard for
shapedvesicleswithanarrowanteriorneckandawiderposterior
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3DStructureofApicalComplexofPsammosa
describing the flagellar apparatus across diverse eukaryotes [15]. TEM and serial ultrathin sections to confirm that P. pacifica
Functionally,theflagellarapparatusinvolvesmultiplerolessuchas possesses a flagellar apparatus comparable with those of dinofla-
flagellarmovement,feedingbehavior,andmicrotubuleorganizing gellates and perkinsids, notably with R1 sensu Moestrup 2000 (or
centers(MTOC)duringcytokinesis.Atthesametime,theflagellar longitudinal microtubular root, LMR) connected to the longitu-
apparatus provides the common ground to understand the dinal basal body, and R4 associated with transverse striated root
homology of cell architecture in distant relatives, especially (TSR) connected to the transverse basal body, and, most
cytoskeletal elements [15]. importantly, that the apical complex is associated with the
Recently, some components of the flagellar apparatus, i.e., microtubule bundle that is connected to R1 via electron dense
striated fibre assemblins [16] and SAS6-like [17] were demon- material. Overall, we demonstrate a connection between the
strated to localize to the apical complex, which strongly suggests apical complex and the flagellar apparatus, and by providing its
that the apical complex evolved from the flagellar apparatus. exact position within the context of the universal flagellar
However, no apicomplexan is known tohave the apical complex apparatus system also provide evidence for the homology of the
and the flagellar apparatus at the same time: the apical complex apicalcomplex tothedinoflagellate peduncle.
exists only in the invasive stages where the flagellar apparatus is
morphologically reduced to a pair of centrioles, and flagella are Results
onlyknowninsomegametesthatlackanapicalcomplex.Sothis
hypothesisisdifficulttotestdirectlyintheapicomplexans.Butthe TEM Tomography and 3D Reconstruction of the Apical
apical complex is not restricted to apicomplexans, and is also Complex
foundinasmallandlittlestudiedcollectionoffree-livingrelatives. Psammosa pacifica is a barley shaped cell with two flagella
Apicomplexans are members of a larger group, the alveolates, subapically inserted in the ventral side throughout the life cycle
which also includes ciliates and dinoflagellates, within which (Fig.1a).Theflagellatecellpossessestheapicalcomplexcomposed
apicomplexans and dinoflagellates are sisters and form a group of microtubules and electron dense vesicles in the apical area
with a handful of lesser-known organisms, collectively called (Fig.1b–c).
myzozoans [18].Myzozoans are characterized by ‘‘myzocytosis’’,
To further investigate the fine structures and spacial relation-
a mode of predation originally described in dinoflagellates [19],
ships of the apical complex and the flagellar apparatus, we
wherethepredatorpokesaholeintheplasmamembraneofaprey
observed two cells by TEM tomography and multiple cells by
celltosuckoutitscytoplasmintoafoodvacuole.Thesepredators
conventional TEM. Figure 2 shows the reconstructed apical
havebeenfoundtouseavariantoftheapicalcomplextomediate
complex overlaid on an illustration of thecelltoshow its relative
myzocytosis[18,20–24]:apicalcomplex-mediatedhostinvasion,it
position.MovieS1showsthetomographicreconstructionand3D
turns out, isavariation ofmyzocytosis intheopposite direction.
model.Theapicalcomplexis,notsurprisingly,locatedattheapex
The‘‘archetype’’apicalcomplexintheselineagesconsistsofan
ofthecell,correspondingtothegulletopening(G)onthelobethat
open-sided conoid, or pseudoconoid [25], and a diversity of
dividestheventralsideofthecelltotheright-anteriorandtheleft-
vesicularcomponents,includingelementsdefinedasrhoptriesand
posterior part. At the anterior end of the pseudoconoid, eight
micronemes, as well as additional membrane-bound structures in
microtubulesformashallowconvexsheetopentotheventralside
some cases [5,6,18,20,21,23,26–33]. In these organisms, any
towards the gullet. In addition to the eight conoid microtubules
association between the apical complex and flagellar apparatus
(CM),thereisalsoabandofextendedconoidmicrotubules(ECM:
can be examined directly, which is what we describe here.
Fig.2).Theconoidmicrotubulesareshortandonlyappearonthe
Previously we described Psammosa pacifica, a new lineage of
dorsal side of theapical complex, and theECM are immediately
predator that branches near the split between apicomplexans
posterior and extend further to the posterior of the cell, towards
and dinoflagellates at the base of the dinoflagellate lineage, and
thebasalbodyandtheflagellarapparatus.TheECMfallbetween
possesses the archetype apical complex with pseudoconoid in the
thegulletandtheotherlongvacuolelocatedontheventralsideof
flagellatedcell[34].TheapicalcomplexofP.pacificaisimportant
the right anterior part of the cell (Fig. 3). Posterior to the apical
not only in the context of the apicomplexans, but also its
complex, the ECM are joined by another set of microtubular
subsequentearlyevolutionindinoflagellates.Dinoflagellateslacka
strands(MS)thataredirectlyconnectedtotheflagellarapparatus
structure readily recognizable as the apical complex,[27–29,31],
(see below). Between the conoid microtubules and the gullet, lie
butithasbeenhypothesizedthatitwascompletelylost,orrather
the micronemes (Fig. 2, 3). Each microneme is clevate, with the
drastically changed in morphology during their early evolution.
narrower anterior neck and the broader, rounded posterior. The
Specifically,somedinoflagellatespossessanintracellularstructure
micronemesarealignedparalleltoeachothertoformtwoclusters:
called peduncle, which is used for myzocytosis [19], and it has
one(Mic1)isassociatedwiththeinsideoftheconvexcurveofthe
been postulated that the peduncle may be homologous to the
pseudoconoid, and another (Mic2) found posterior to the first
apical complex [18,27] based on its function and the presence of
cluster. There are no ultrastructurally recognizable difference
the peduncle-associated microtubular basket/strands, which are
between themicronemesof Mic1andMic2.
hypothesized to be homologous to the microtubular apical
Interestingly, the second cell that we observed by serial TEM
complex [35–39].
tomography had a mature apical complex, and also a small
To resolve these issues in early apical complex evolution, we
nascent apical complex (Fig. 4; Movie S2 for tomographic
used serial TEM tomography for the first time to reconstruct the
reconstruction and 3D model). The nascent pseudoconoid (C’) is
3D structure of an apical complex, that of P. pacifica, and to
located left to the mature pseudoconoid (C) and the conoid
directly examine its association with the flagellar apparatus. The
microtubulesofthenascentpseudoconoidarerightangletothose
resulting3Dmodelshows:(1)thedetailsofitseight-microtubular
pseudoconoid,withadditionalmicrotubulesrunninglongitudinal- of the mature pseudoconoid. There are four clusters of micro-
ly towards the flagellar apparatus, (2) two clusters of microneme- nemes (Mic1, Mic2, Mic19, Mic29) rather than two, altogether
like organelles associated to the microtubular pseudoconoid, and consistent with the conclusion that this cell is in the process of
(3) an association between the apical complex and a ‘‘gullet’’ duplicatingitsapicalcomplex,whichisnormallycomposedofthe
positioned at the cell apex. In addition, we used conventional pseudoconoid and twoclustersof micronemes.
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Figure1.GeneralmorphologyofPsammosapacifica.a.SurfacestructureoftheventralsideofPsammosapacifica,showingtwoflagellainserted
subapicalregionoftheventralside.Theopeningofthegullet(arrowhead)islocatedatthecellapex.b.Alongitudinalsectionofthecellshowinga
clusterofmicronemes(M),densevesicles(d)andthenucleus(N).c.Thelongitudinalsectionoftheapicalregionshowsaclusterofmicronemes(M),
densevesicles(D)betweentheopeningofthegullet(arrowhead)andamitochondrion(mit).Singlemembraneboundtrichocysts(T)arelocatednear
theclusterofmicronemes.Alveolivesicles(asterisk)areabsentatthegulletandtrichocysts.N:nucleus.Scales:5mmina,1mminb,500nminc.
doi:10.1371/journal.pone.0084653.g001
Flagellar Apparatus transverse basal body (TB) has an electron dense collar near the
TheoverallfeaturesoftheflagellarapparatusoftheP.pacificais transition region.
comparable to that of the dinoflagellates. In this article, we Root1(R1)sensuMoestrup2000iscomposedofmorethan25
primarily adhere to the universal numbering system of Moestrup microtubules, of which 15 microtubules are connected to the
2000,supplementedbythetraditionaldinoflagellateterminology, dorsal side of the longitudinal basal body (Fig. 6). The proximal
assubstantialstudieshavebeendonebeforethestandardizationof end of R1is lined withan electrondense sheet. Thin connecting
theterminology of flagellar apparatus components[38]. fibersjointheelectrondensesheetandthetransversebasalbody.
Figure 5 illustrates the main components of the flagellar The proximal end of the approximately ten remaining root 1
apparatus and connections between the pseudoconoid microtu- microtubules(R1’)separatefromthelongitudinalbasalbody(LB)
bules,theextendedconoidmicrotubules(ECM),andtheflagellar and are connected tothe wide band of microtubule strands (MS)
apparatus. The transverse and longitudinal basal bodies are viaelectrondensematerialsattheposteriorregionoftheproximal
inserted sub-apically at nearly right angle to one other on the endofthebasalbody.TheMSbypasstheventralsideofthebasal
ventralsideofthecell(Fig.3,6,7).Basalbodiesareconnectedby bodyalongtheridgeoftherightanteriorpartofthebodybetween
six strands of fibrous connective material on the dorsal side. The the dorsally located gullet and the ventrally located vacuole, and
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3DStructureofApicalComplexofPsammosa
Figure2.TomographicreconstructionofthePsammosapacificaapicalcomplex(Cell1).ThemicrotubulecomponentoftheP.pacifica
apicalcomplexiscomposedofeightshortconoidmicrotubules(C,red)andasimilarnumberofextendedconoidmicrotubules(ECM,brown)thatare
longerandextendtowardstheposteriorofthecell.Thevesicularcomponentsincludestwoclustersofrhoptries;oneofwhich(M1,turquoise)ison
theventralsideofthepseudoconoid,andtheother(M2,green)isontherightsideofECM.Italsoincludesthelargegullet(G,purple)andseveral
sphericaldensegranules(D,blue).
doi:10.1371/journal.pone.0084653.g002
join the ECM (Fig. 3). There are six short microtubules on the (anteriorofthecell)andthehypocone(theposterior).Thecellhas
dorsalsideofLBtoformroot2(R2),whichareconnectedtoTB, twoflagellainsertedonthedorsalsideofthejunctionbetweenthe
LBandtheelectrondensesheetofR1(Fig.6;arrowheads).Root3 epiconeandthehypocone.Indinoflagellates,therearetwoeasily
is missing in P. pacifica, and Root 4 (R4) is single microtubule recognizableflagellarroots;root1(R1),whichisconnectedtothe
associatedwithtransversestriatedroot(TSR)(Fig.3,7,8).R4and ventral side of longitudinal basal body, and root 4 (R4), which is
the TSR are connected to the dorsal side of the transverse basal accompanied by the transverse striated root (TSR) and is
body by fibrous connective material (Fig. 7). This root extends connected to the dorsal side of the transversal basal body. In
verticallytocrosstheleftanteriorpartofthecell(Fig.8).Thereis additiontothesetworoots,theyhaveasinglemicrotubularroot3
noobviousconnectionbetweenR4andsurfacemicrotubulesthat (R3) root that is connected to the ventral side of the transverse
are located beneath thealveolivesicles (Fig.3). basal body. In some species, a single microtubular root 2 (R2) is
also present and connected to the dorsal side of the longitudinal
Discussion basal body. Other notable features are ‘‘collars’’ around basal
bodies. In most dinokaryotes, both longitudinal and transverse
ComparisonoftheFlagellarApparatusacrossMyzozoans
basalbodyareaccompaniedbyastriatedfibrousstructure.Collars
Figure9summarizestheorganizationoftheflagellarapparatus
and the fibers between them (often striated) contain centrin
and the apical complex for characterized myzozoans. Among
molecules, and are hypothesized to be involved in the flagellar
myzozoans, the ultrastructure of the flagellar apparatus of the
movement [40].
dinoflagellate is well established (reviewed in [14,38,40,41]).
Dinoflagellate cells are comprised of two subregions: the epicone
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Figure3.SerialTEMoftheextendedconoidmicrotubules(ECM)andmicrotubularstrands.a–g.Excerptsfromaseriesofserialsections
proceedingfromtheventraltothedorsal.Theextendedconoidmicrotubules(ECM)arealignedbetweentoshortconoidmicrotubules(CM)and
microtubulestrands(MS),andextendtotheposteriortowardsthebasalbodies,followingthelineofthegullet(G)andalargeelongatedvesicle.
Arrowhead:theboundarybetweenCMandECM.Arrow:theboundarybetweenECMandMS.Scalebars=500nm.
doi:10.1371/journal.pone.0084653.g003
Oxyrrhis marina isone of the basal lineages of dinoflagellates. O. shortened,R4ispresentwithTSRandR1retainstheconnection
marina lacks the microtubular strands/basket, perhaps because O. tothepseudoconoidviatheextendedconoidmicrotubules(ECM).
marina does not feed by myzocytosis, but rather by phagocytosis The extension of R1 and the ECM in Psammosa is most likely
using a bulging structure called ‘‘tentacle’’ located near the homologous to the bypassing MS of the dinoflagellate peduncle,
flagellar insertion. Here, an additional V-root emerges on the basedonthespacialrelationshipstothebasalbodiesinthecell.In
ventral side of the longitudinal basal body and extends into the dinoflagellates,theMSisassociatedwiththepeduncle,thoughthe
tentacle, where numerous profiles of electron dense vesicular orientation of the peduncle in relation to the microtubular
structure have been observed [42,43]. Elongated electron dense strands/basket varies among species; the peduncle emerges
vesicles were also reported fromAmoebophrya, a parasitic flagellate anteriortothebasalbodiesinsomecases[36,45],whileinothers
that belongs to a basal dinoflagellate lineage, syndinians [44]. A it emergesposterior [35,37,38,46,47].
possible association between the microtubular basket and the Except for Perkinsus and Psammosa, dinoflagellates lack a
flagellar apparatus remains unknown in Amoebophrya, since the structure readily recognizable as the apical complex. The
short-lived invasive stage lacks flagella or a complex flagellar functional similarity but the weak morphological resemblance
apparatus, analogous totheapicomplexans. between the apical complex and the dinoflagellate peduncle has
The microtubular roots of P. pacifica can be viewed as a been noted [18,25,27]. However, their homology has been a
variation of those of the dinoflagellates: R3 is absent, R2 is subject of debate [48]. The demonstration here of a physical
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Figure4.TomographicreconstructionofmatureandnascentapicalcomplexesofP.Pacifica(Cell2).Inthesecondcellwheretheapical
complex was reconstructed by serial tomography, two sets of apical complexes were found, one large and mature, and a second smaller. The
structuresobservedareasinFigure1,butineachcasethenumberisdoubled,sotherearetwopseudoconoidsconsistingofeightCMs,twoECMs,
fourclustersofmicronemes,andseveralsphericaldensegranules.AllabbreviationsareasinFigure1.
doi:10.1371/journal.pone.0084653.g004
connectionbetweentheflagellarapparatusandtheapicalcomplex perkinsid apical complex is also physically linked to its flagellar
in Psammosa via R1+ECM strongly supports the homology apparatus.
between the dinoflagellate peduncle and the apical complex. In Colpodella vorax, a flagellate thought to be closer to the
Dinoflagellates, perkinsids, colpodellids and Psammosa all have a apicomplexans where the apical complex and flagellar apparatus
bundleofmicrotubulesthatbypassthedorsalsideofbasalbodies arefoundsimultaneously[20],twomicrotubularroots,R1(‘‘pR’’)
fromtheright posterior tothe leftanterior of thecell. and R4 (‘‘aR’’) are associated with the posterior basal body and
Amongperkinsids,additionalmicrotubulesthatbypassthebasal the anterior basal body, respectively. In addition, there is a
bodies, split into two strands, and extend anteriorly towards the microtubular strand (‘‘oR’’) obliquely passing the flagellar
apical complex have been reported in Parvilucifera infectans [27]. apparatus.R4extendstotheanteriorapex,beyondtheareawith
However,aconnectionbetweentheseadditionalmicrotubulesand subsurface pellicular microtubules, and locate near the open side
the apical complex was not reported. In Perkinsus marinus, on the of theC-shapedpseudoconoid [20].
other hand, a few microtubules of the pseudoconoid extends Interestingly, during the course of our work, Portman etal.
posteriorly toward the flagellar apparatus, but again a possible reportedasimilarmicrotubularextensionneartheapicalcomplex
connectionofthesemicrotubulestotheflagellarapparatuswasnot of Chromera velia [49], a photosynthetic relative of apicomplexans.
reported [30,31].Taken together withour results, wesuggest the In this report, one view (their figure 4B–E) shows the bypassing
microtubules (as opposed to the description in the text, which
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3DStructureofApicalComplexofPsammosa
Figure5.OverviewoftheapicalcomplexandflagellarapparatusofP.pacifica.Dorsalview.b.Ventralview.Thepseudoconoid(C)consists
ofeightconoidmicrotubulesandabandofextendedconoidmicrotubules(ECM)thatarepositionedontheventralsideofthegulletandextend
posteriorlytowardsthebasalbody.TheECMmeetthemicrotubularstrands(MS)thatbypasstheventralsideofthebasalbodiesalongtheridgeof
rightanteriorpartofthecell.TheMSisconnectedtothe6ventralmicrotubulesoftheR1/LMR.R1/LMRislinedwithafibroussheetonthedorsalside
andisconnectedtothelongitudinalbasalbody(LB).Afibrousconnective(dorsalrootconnective:DRC)materialontheleftsideofLBconnecttothe
posteriorsideoftransversebasalbody(TB).TheLBandTBareatapproximatelyrightanglestooneanother.TBhasafibrous‘‘collar’’(transversebasal
bodycollar:TBC)ofpartialdonutshape.
doi:10.1371/journal.pone.0084653.g005
claims that these microtubules terminate at the anterior basal ReplicationoftheApicalComplexandPossiblePresence
body) that meet the apical complex at a right angle. These of the Apical MTOC in Psammosa
microtubulesprobablycorrespondtothepreviousobservationofa
The second Psammosa cell that we observed by tomography
‘‘double layered pseudoconoid’’ by Oborn´ık etal (Fig. 36, 38 in
showed the duplicated clusters of micronemes and the nascent
[50]). It is unclear if the posterior end of the bypassing
pseudoconoid. The principle components of the two apical
microtubules(unannotatedintheirfigure4B)hasanyconnection
complexes are the same; the eight-microtubular pseudoconoid
to R1, as we show in Psammosa and is known in dinoflagellates.
andtwoclustersofmicronemes.Thenascentapicalcomplexisnot
TheyalsoshowR4extendingfromthedorsalsideoftheanterior
connected to the ECM, which suggests the duplication of the
flagellum, which was originally interpreted to show that ‘‘part of
pseudoconoidmicrotubulesareformedattheapex.Inthecaseof
the subpellicular microtubule array lies alongside the anterior
T. gondii, an independent ring-shaped MTOC for subpelicullar
flagellar groove’’. R4 is shown extending near the pseudoconoid,
microtubules, named the apical polar ring (APR) exists at the
analogoustoC.vorax[20].R2issimilartothatofPsammosainthat
anterior oftheapicalcomplex[51,52,53].Itwouldbeinteresting
itconsistsofshortmicrotubulesthatliebetweentheposteriorand
toknowifPsammosahasasimilarapicalMTOCforpseudoconoid
theanterior basal bodies.
formation. Psammosa cells replicate by horizontal binary division
[34], identical to that of Oxyrrhis marinus, which has a subsurface
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3DStructureofApicalComplexofPsammosa
Figure6.Longitudinalbasalbodyandassociatedstructures.a–h.seriesofnearlytransversalserialsections,proceedingfromanteriorto
posterior. Arrowheads: dorsal root connective material. Asterisk: thin connective fibers. L: longitudinal basal body. MS: microtubular strands. R1:
microtubularroot1,whichisalsocalledthelongitudinalmicrotubularroot(LMR).R1’:apartofR1thatconnectstoMS.R2:microtubularroot2,which
iscomposedof6veryshortmicrotubules.T:transversebasalbody.Arrowhead:thefibrousconnectivebetweenMSandR1.Theinsetinpanel(a)
showstherelationshipoftheplaneofthesesectionstothecell.Scalebars=500nm.
doi:10.1371/journal.pone.0084653.g006
ringMTOCareaonthedivisionplane[54].Inaddition,Psammosa eveninheritanceoftheorganellestodaughtercellsduringdivision.
must have an MTOC for the basal body and flagellar root In Psammosa, the apical complex is directly connected to the
duplication. longitudinalbasal body throughseries ofconnectedmicrotubular
structures; ECM, MS, and R1, which are connected to one
The Origin and Evolution of the Apical Complex another via fibrous connections, none of which is striated. In
Emergingevidencebasednotonlyonthemorphologybutalso ColpodellavoraxandChromeravelia,R4isclosetotheapicalcomplex,
the molecular composition suggests the apical complex and the though not directly connected. R4 in Psammosa, dinoflagellates,
flagellar apparatus are connected. Francia etal [16] recently and perkinsids has a striated fibre, though in Colpodella and
reportedthatanon-microtubular,fibrousconnectionbetweenthe Chromera,theassociatedfibredoesnothavestriation.Itwouldbeof
apical complex of T. gondii and the MTOC contains homologues interest to know if R1 or R4 associated fibers contain SF-
of SF-assemblin, one of the components of flagellar apparatus assemblin.
foundingreenalgae/plantsandastramenopileprotists[55,56].In The SAS-6 like protein (SAS6L) [17] is another interesting
greenalgae,SF-assemblinislocalizedatthestriatedmicrotubule- candidate for the apical complex-flagellar apparatus connection.
associatedfiber(SMAF)associatedwiththemicrotubularflagellar SAS6LislocalizedatthepreconoidalringofT.gondiitachyzoites,
roots [57,58]. In T. gondii, the fibrous structure that contains SF- whereas located at the basal plate of the flagella in Trypanosoma
assemblin homologues does not show striated pattern, but still brucei.TheexactfunctionofSAS6Lremainsenigmaticandisnot
maintains theconnection between the inner microtubular pair of essentialintachyzoites.Ifitisinvolvedinbasalbodyformation,as
the apical complex and the centrioles, and therefore ensures the issuggestedinTr.brucei,SAS6Lmighthaveaprimaryroleinthe
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3DStructureofApicalComplexofPsammosa
Figure7.Transversalbasalbodyandassociatedstructures.a–f.Aseriesofnearlytransversalserialsections,proceedingfromanteriorto
posterior. MS: microtubular strands. R4: microtubular root 4. T: transverse basal body. TC: transverse collar. TSR: transverse striated root. Scale
bars=500nm.
doi:10.1371/journal.pone.0084653.g007
other stages of T. gondii such as in microgametes flagellar The majority of apical complexes examined so far have both
formation. The location of any SAS6L homologues in Psammosa rhoptries and micronemes, and, in perkinsids, also conoid
would also be interesting in the context of MTOC and associated micronemes (CAM). However, in several cases one or
microtubular populations. moreofthesestructuresisabsent,solosingvesicularcomponents
Figure 10 shows a reconstruction of character evolution of the of the apical complex appears to be common. For example,
apical complex. The apical complex traces back to the common Haemogregarina magnaretains only micronemes[59],Cryptosporidium
ancestoroftheapicomplexanparasitesandthedinoflagellates,i.e. sp.exRuditapesdecussatusretainsonlyrhoptries[60],andColpodella
an ancestral myzozoan. The ‘‘archetype’’ apical complex is pugnax retains only rhoptries [23]. These are scattered across the
composed of a pseudoconoid, or open-sided conoid [25], which different lineages of myzozoans, suggesting several independent
arestillfoundamongcolpodellids,perkinsids,andPsammosa.Inthe losses. Indeed, the apicomplexan Theileria parva changes its
apicomplexan parasites, the conoid developed the truncated vesicularcomponentsdependingonlifestage-specifichostinvasion
conical shape with a pair of intra-conoidal microtubules seen strategies; sporozoites and merozoites lack micronemes, whereas
today,andthepolarringwasaddedtotheanteriorend.Chromera ookinetesretainbothrhoptriesandmicronemes(forreview,[61]).
velia also is an interesting intermediate variant, in that the In the apicomplexans, rhoptry proteins are injected into the host
pseudoconoid is open-sided, but with a pair of intra-conoidal cell,whereasmicronemeproteinsaresecretedonthesurfaceofthe
microtubules and the additional layer of bypassing microtubules parasite itself to mediate the host-parasite interaction [62].
[49,50]. It is unclear if either layer of the microtubules are Psammosa possesses a single type of elongated, rhomboid shaped
connectedtotheothercytoskeletalelementsinC.veila,andfurther vesicle, which we referred to as micronemes as our preliminary
study isneeded.In thedinoflagellates, thestructure ismostlikely investigation of Psammosa EST includes genes with conserved
developed topeduncle asdiscussed above. domains that are found in microneme proteins in various
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3DStructureofApicalComplexofPsammosa
Figure8.Root4andtransversestriatedroot(TSR).a–c.Aseriesofnearlylongitudunalserialsections,priceedingfromventraltodorsal.M:
mitochondrion. MS: microtubular strands. N: nucleus. R1: microtubular root 1. R4: microtubular root 4. TF: transverse flagellum. TSR: transverse
striatedroot.Scalebars=500nm.
doi:10.1371/journal.pone.0084653.g008
apicomplexans (unpublished data). As a predator, P. pacifica does andapicomplexansclearlyprecededtheselingeages[65],suggest-
not require the same interactions with its prey, so this may have ing the ancestral myzozoan was most likely either an endosym-
ledtoareductionofitsvesicularcomplexity.Conversely,however, biont or a mixotroph, so parasitism based on the apical complex
the apical complex of P. pacifica is unique in that it is associated probably evolvedmuch later.
with the gullet, a permanent invagination at the apex of the cell,
andalargevacuole.InChromera,alargevacuole,whichisapartof Concluding Remarks
theendomembranesystemsituatedposteriortothepseudoconoid, We reconstructed the three dimensional structure of the
though without an openingas foundin Psammosa[49]. apical complex and flagellar apparatus of the basal sister to
dinoflagellates, P. pacifica. This provides the first demonstration
Myzocytosis and the Origin of Parasitism of a direct microtubular link between these two cytoskeletal
The function of the apical complex is perceived primarily as structures. Based on an ultrastructural comparison of the
invasion, but myzozoans include lineages of diverse trophic flagellar apparatus and associated structures across a wide
strategies: parasitism, predation, photoautotrophy, and mixotro- range of alveolates, we also conclude that the peduncle, a
phy. Not only parasites, but also free-living grazers and feeding apparatus found in myzocytotic dinokaryotes, is
photosynthetic algae retain the apical complex, obviously for a probably homologous to the apical complex. Further under-
variety of functions. In predatory and mixotrophic lineages, the standing of the apical complex of P. pacifica at the molecular
apical complex is used for myzocytotic feeding. In C. velia, a level, as well as the investigation of the 3D structure of the
photosyntheticrelativeoftheapicomplexansthatisthoughttobe apical complex in related lineages and molecular evidence for
acoralendosymbiont[63],theapicalcomplexmayhavearolein proteins shared between the apical complex and the flagellar
establishmentofendosymbiosis[64].Theplastidofdinoflagellates apparatus will all elucidate the relationship between and
PLOSONE | www.plosone.org 10 January2014 | Volume 9 | Issue 1 | e84653