characterization of the subpellicular network, a filamentous membrane skeletal component in the...

Molecular & Biochemical Parasitology 115 (2001) 257–268

Characterization of the subpellicular network, a filamentousmembrane skeletal component in the parasite Toxoplasma gondii�

Tara Mann b, Con Beckers a,b,*a Di�ision of Geographic Medicine, Uni�ersity of Alabama at Birmingham, Birmingham, AL 35294-2170, USA

b Department of Cell Biology, Uni�ersity of Alabama at Birmingham, BBRB 540, 845 19th Street South, Birmingham, AL 35294-2170, USA

Received 30 January 2001; received in revised form 19 April 2001; accepted 20 April 2001

Abstract

Electron microscopic examination of detergent-extracted Toxoplasma tachyzoites reveals the presence of a mechanically stablecytoskeletal structure associated with the pellicle of this parasite. This structure, composed of interwoven 8–10 nm filaments, isassociated with the cytoplasmic face of the pellicle and surrounds the microtubule-based cytoskeleton. Two protein componentsof this network, TgIMC1 and TgIMC2, were identified. Both are novel proteins, but have a resemblance to mammalian filamentproteins in that they are predicted to have extended, coiled-coil domains. TgIMC1 is also homologous to articulins, the majorcomponents of the membrane skeleton of algae and free-living protists. A homologue of TgIMC1 in the related malaria parasitePlasmodium falciparum was also identified suggesting the presence of structurally similar membrane skeletons in all apicomplexanparasites. We suggest that the subpellicular network, formed by TgIMC1 and 2 in Toxoplasma gondii and related parasites, playsa role in the determination of cell shape and is a source of mechanical strength. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Toxoplasma gondii ; Cytoskeleton; Membrane skeleton

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1. Introduction

The protozoan Toxoplasma gondii is an obligate in-tracellular parasite that is able to infect and thrive inmost nucleated cells of warm-blooded animals [1,2]. Inhumans and a variety of animal species this parasitecan cause severe infections, especially when the immunesystem is not fully developed or is seriously compro-mised in any manner [3,4]. During its normal life cycle,Toxoplasma needs to withstand a variety of physicallyand chemically stressful environments, such as thoseencountered in the circulatory system and the gas-trointestinal tract of the host organism, as well as

outside the host. In addition, the parasite is deformeddramatically during the invasion of host cells [5–8]. Inorder to maintain structural integrity under these con-ditions, Toxoplasma needs a source of mechanicalstrength.

Mechanical stability of Toxoplasma and related api-complexan parasites is most likely dependent on thepellicle and its underlying cytoskeletal components. Thepellicle is a three-unit membrane composed of theplasma membrane and a system of flattened vesiclesknown as the inner membrane complex. The only cy-toskeletal structures known to be associated with theToxoplasma pellicle at this time are the 22 subpellicularmicrotubules. These originate from an apical structure,the polar ring, and run two-thirds down the length ofthe parasite in a spiraling fashion [9–11]. Based onstudies of other protists with cortical microtubule-basedcytoskeletons, such as the Euglenoids and Try-panosomes, the subpellicular microtubules are likely tobe associated with the pellicle through an interactionwith proteins embedded in or bound to the pellicularmembranes [12,13]. Freeze fracture analysis of suchprotists has, in fact, revealed the presence of intramem-

Abbre�iations: CC, putative coiled-coil domain; CRD, cysteine-richdomain; DOC, sodium deoxycholate; IPTG, isopropyl-�-D-thiogalac-toside; PBS, phosphate buffered saline; TBST, Tris-buffered salinecontaining Tween20.

� Note: Nucleotide sequence data reported in this paper are avail-able in the GenBank™, EMBL and DDBJ databases under theaccession number AY032678 for IMC1 and AY032682 for IMC2.

* Corresponding author. Tel.: +1-205-9341633; fax: +1-205-9345600.

E-mail address: [email protected] (C. Beckers).

0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 6 -6851 (01 )00289 -4

T. Mann, C. Beckers / Molecular & Biochemical Parasitology 115 (2001) 257–268258

branous particles (IMPs) in the plasma membrane andthat these are distributed in a manner suggestive of adirect association with the cortical microtubules. Freezefracture studies of Toxoplasma and related apicom-plexan parasites have also revealed regular arrays ofintramembranous particles (IMPs) on the inner andmiddle faces of the inner membrane complex. TheseIMPs are uniform in size and are arranged in a patternof spiraling longitudinal single and double rows with a32 nm periodicity. It has been suggested that the doublerows of IMPs are associated with the subpellicularmicrotubules. Considering, however, that all IMP rowsextend along the entire length of the parasite, even theposterior regions where microtubules are absent, anadditional filament system must be present that is orga-nized with a 32 nm periodicity and extends along theentire length of the parasite [14–16]. In the relatedapicomplexan parasites Sarcocystis and Besnoitia, apotential candidate for such a structure, a filamentousnetwork of unknown composition and localization withrespect to the pellicle, has been observed [17]. Althoughsuch a structure has not been described in Toxoplasmathus far, it is possible that the single row IMPs inToxoplasma are associated with a similar network.

The subpellicular microtubules are unlikely to be amajor source of mechanical strength for the parasites asthey extend only two-thirds along the length of theorganism. Furthermore, their disruption with dini-troanilide herbicides does not appear to affect the para-sites’ normal resistance to cell disruption procedures[16]. This would then leave the pellicular membranes or,possibly, a pellicle-associated membrane skeleton as themain elements that maintain the structural integrity ofToxoplasma.

We have identified a new cytoskeletal element inToxoplasma gondii that we have named the subpellicu-lar network. This structure is composed of interwovenfilaments and extends from the polar ring along theentire length of the parasite. The network is located onthe cytoplasmic face of the inner membrane complex,where it appears to form a membrane skeleton. Inaddition, we also report on the molecular characteriza-tion of two major protein subunits of this structure,TgIMC1 and TgIMC2.

2. Materials and methods

2.1. Materials

All chemical reagents were obtained from FisherScientific (Pittsburgh, PA) unless otherwise noted. GailJohnson (University of Alabama at Birmingham, Birm-ingham, AL) kindly provided the monoclonal �-tubulinantibody, 5H1, used in immunofluorescenceexperiments.

2.2. Culture of parasites

The RH (HXGPRT−) strain of T. gondii (kindlyprovided by Dr David Roos, University of Pennsylva-nia) was maintained by serial passage in confluentcultures of human foreskin fibroblasts (HFF) or VEROcells in �-minimal essential medium (Mediatech, Hern-don, VA) supplemented with 10% fetal bovine serum(Hyclone, Logan, UT), 2 mM glutamine, 100 IU ml−1

penicillin, and 100 �g ml−1 streptomycin. Parasiteswere harvested by syringe passage followed by centrifu-gation at 2000 rpm for 5 min.

Asynchronous cultures of Plasmodium falciparuminfected erythrocytes used for immunofluorescenceor immunoblotting were provided by Dr NaomiLang-Unnasch (University of Alabama at Birming-ham).

2.3. Isolation of cytoskeletons and production ofantisera

All manipulations were performed at 0–4°C. Extra-cellular parasites were resuspended in PBS containing1% Triton X-100 (TX-100) or 1% sodium deoxy-cholate (DOC) at a concentration of 1–5×107 para-sites ml−1, and homogenized by 20–40 strokesin a Dounce homogenizer, nitrogen cavitation or soni-cation. Samples were layered on a cushion of 15%glycerol in either 1% TX-100 or DOC in PBS andcentrifuged for 10 min at 10 000 rpm. The supernatantwas carefully aspirated and the pellet resuspended inPBS and examined by electron microscopy as describedbelow.

For the production of antisera to cytoskeletalproteins, 4×109 parasites were extracted in 1% DOC inPBS and purified as described above. The pellet wasresuspended in PBS and used to immunize two mice bysubcutaneous injection (Cocalico Biologicals, Ream-stown, PA). After 3 weeks, a booster injection wasgiven with a similar quantity of antigen.

2.4. Library screening

A T. gondii cDNA library in �ZAPII (AIDS Re-search and Reference Reagent Program, McKessonBiosciences, Rockville, MD) was screened with anti-cy-toskeleton antisera using the ProtoBlot Immunoscreen-ing System (Promega). Positive plaques were isolatedand re-screened using the same method. In vivo exci-sion of pBluescript SK phagemids from positive cloneswas performed as described by the manufacturer(Stratagene, La Jolla, CA). Positive clones were initiallygrouped based on restriction enzyme digestion patterns.Plasmid DNA was purified from a single representativeof each group using the Qiagen midi-prep system (Qia-gen, Valencia, CA) and the insert sequenced (Keck

T. Mann, C. Beckers / Molecular & Biochemical Parasitology 115 (2001) 257–268 259

Biotechnology Resource Laboratory, Yale University,New Haven, CT).

DNA and predicted protein sequences were analyzedusing BLAST [18] (http://www.ncbi.nlm.nih.gov) andFASTA [19] (http://www.genome.ad.jp/) programs.Coiled-coil predictions [20] were performed using theCOILS program (www.expasy.ch).

Sequence data for P. falciparum chromosomes(1,3,4,5,6,7,8,9,13) was obtained from The Sanger Cen-tre website at http://www.sanger.ac.uk/Projects/P– falci-parum/. Sequencing of P. falciparum chromosome(1,3,4,5,6,7,8,9,13) was accomplished as part of theMalaria Genome Project with support by The Well-come Trust.

2.5. Preparation of monospecific antisera torecombinant TgIMC1 and TgIMC2

For the production of monospecific antisera toTgIMC1 and TgIMC2, fusion proteins containing por-tions of the respective open reading frames were ex-pressed in Escherichia coli.

For TgIMC1, an insert encoding amino acids 140–610 was excised from a cDNA clone in pBluescriptSKII using Bam HI and Xho I and inserted in thepRSETB vector (Invitrogen, San Diego, CA). Thisconstruct was transformed into JM109 cells and usedfor expression as suggested by the manufacturer. Thefusion protein was purified on Ni-NTA-agarose (Qia-gen, Valencia, CA) and fractions containing pureprotein were dialyzed overnight against PBS at 4°C.The dialysate was centrifuged for 15 min at 15 000×gand the protein in the supernatant was used for immu-nization of mice and rabbits.

TgIMC2 could only be stably expressed in E. coli asa fusion protein with glutathione-S-transferase.An insert encoding amino acids 447–1030 was excisedfrom a cDNA clone in pBluescript IISK in two seg-ments using Bam HI and Nhe I, and Nhe I andEco RI. These were inserted between the Bam HI andEco RI sites of the pGEX1 vector (Amersham Pharma-cia Biotech, Piscataway, NJ) and transformed intoJM109 cells. Expression was carried out as suggested bythe manufacturer. The resulting fusion protein wasinsoluble. The insoluble material was dissolved in 8 Murea in 50 mM Tris–HCl pH 8.0. Any remaininginsoluble material was removed by centrifugation andthe supernatant was dialyzed against overnight againstPBS. The dialysate was clarified by centrifugation andthe GST-IMC2 fusion protein was purified from thesupernatant by preparative gel electrophoresis and elec-troelution (Bio-Rad Laboratories, Hercules, CA).Purified protein was used for the production ofmonospecific antisera in mice and rabbits as describedabove.

2.6. Immunofluorescence

Extracellular parasites in PBS were allowed to adhereto poly-L-lysine coated glass coverslips for 15 min atroom temperature and subsequently washed to removeunbound parasites. Attached parasites were extracted 5min in 1% DOC in PBS, washed three times in PBS,and fixed for 15 min in 3% paraformaldehyde in PBS.Samples were incubated 30 min in primary antibodiesdiluted 1:500 (5H1) or 1:1000 (anti-CYT, anti-IMC1and anti-IMC2) in 3% bovine serum albumin (BSA) inPBS. Bound antibodies were visualized using fluores-cein or rhodamine-conjugated secondary antibodies atthe dilutions suggested by the manufacturer (Bio-Radlaboratories). The coverslips were mounted inMOWIOL. Epifluorescence microscopy was performedusing an Olympus BX60 and images were pho-tographed using Kodak Tmax400 film. Individual nega-tives were scanned using a Nikon LS-2000 scanner(Nikon, Melville, NY) and images processed in AdobePhotoshop (Adobe Photosystems, San Jose, CA).

2.7. Electron microscopy

For examination by transmission electron mi-croscopy, isolated cytoskeletons were prepared as de-scribed above and re-sedimented. The pellet was fixedin 1% glutaraldehyde for 20 min at 4°C and washedonce in PBS. This was followed 20 min incubations in1% tannic acid and subsequently 1% osmium tetroxide,separated by a wash in PBS. After dehydration inincreasing concentrations of ethanol, the samples wereembedded in Spurr’s resin (Electron Microscopy Sci-ences, Fort Washington, PA) and polymerizedovernight at 70°C. Thin sections obtained were stainedwith uranyl acetate and lead citrate and observed in aHitachi H700 electron microscope.

For negative staining experiments, extracted parasiteswere allowed to settle onto carbon-coated copper gridsfor 1 min. Excess liquid was removed and the samplewas stained for 30 s with 2% phosphotungstic acid, pH6.8. After a brief wash in distilled water, the sample wasexamined by electron microscopy.

For metal-shadowing, extracted parasites were al-lowed to settle onto carbon-coated copper grids anddehydrated in increasing concentrations of ethanol.Standard critical point drying was performed in a Balz-ers CPD 020 (Balzers, Hudson, NH) and compared todrying by evaporation after treatment with hexamethyl-disilazane (HMDS) [21]. Briefly, the sample was firstincubated in HMDS at a dilution of 1:1 with 100%ethanol for 5 min followed by 5 min in HMDS alone.The sample was then allowed to air dry and sputtercoated (EMScope SC500, Electron Microscopy Sci-ences, Fort Washington, PA) with gold/palladium at 10mV for 30 s. We found the methods to yield compara-


ble results and used the HMDS technique for all subse-quent experiments.

2.8. Immunoelectron microscopy

For pre-embedding immunoelectron microscopy, acrude pellicle preparation was isolated. Free parasites at5–7×107 ml−1 in PBS were broken using a BransonSonifier 250 (Branson, Danbury, CT) equipped with amicrotip by sonication for 10 s at a 50% duty cycle. Thepellicle fraction was collected by centrifugation at10 000 rpm for 10 min and washed in PBS. Isolatedpellicles were incubated with primary antibody (anti-TgIMC1 at 1:1000 or anti-TgIMC2 at 1:500) diluted in3% BSA in PBS for 2 h on ice. The sample was washedin PBS by sedimentation (10 000 rpm for 10 min) andresuspension. Incubation with the IgG goat anti-mousesecondary 10 nm gold conjugate (Sigma, St. Louis,MO) diluted 1:25 in 3% BSA in PBS was performedovernight at 4°C. The sample was then washed in PBSas described above and fixed in 1% glutaraldehyde inPBS for 20 min. After washing the sample was thenfixed in 1% osmium tetroxide in PBS for 20 min. Theembedding protocol was as described above.

For immunolabeling of metal shadowed samples, theisolated pellicles or detergent-extracted parasites wereallowed to settle onto the formvar-coated grids. Allincubation were performed by inverting the grids on to40 �l drops of solution and washed three times in PBSbetween each step. The grids were first blocked in 3%BSA in PBS for 5 min and then incubated for 1 h atroom temperature in a humid chamber with primaryantibody (anti-TgIMC1 diluted 1:1000 or anti-TgIMC2diluted 1:250) diluted in 3% BSA in PBS. Samples werethen incubated for 1 h at room temperature in humidchamber with a 1:25 dilution IgG goat anti-mouse 10nm gold conjugate. The samples were fixed in 1%glutaraldehyde in PBS for 20 min. Dehydration andshadowing was performed as described above.

2.9. SDS-PAGE and immuno blotting

Protein preparations were separated by SDS-PAGEon 12% polyacrylamide gels as described [22]. Whereindicated, proteins were transferred to nitrocelluloseand probed with different antisera as described before[23]. Bound antibodies were detected using the SuperSignal kit from (Pierce, Rockford, IL).

3. Results

3.1. Isolation of Toxoplasma cytoskeletons

In an attempt to understand the basis for the me-chanical stability of T. gondii, we decided to character-

ize the protein composition of isolated parasitecytoskeletons. To this end, we set out to develop aprotocol that would allow for the isolation of cytoskele-tal preparations with a sufficient yield and degree ofpurity for the development of antisera. Our initialattempt used a procedure developed by Nichols et al.that was based on extraction with the non-ionic deter-gent Triton X-100 in combination with cell homoge-nization [9]. As judged by negative staining electronmicroscopy, the resulting preparations appeared to bepoorly extracted with only 5–10% of all parasites suffi-ciently extracted for visualization of the cytoskeletalelements by negative staining (Fig. 1A). Further analy-sis of these preparations by thin section electron mi-croscopy confirmed this result and revealed that theinner membrane complex in particular seems to beresistant to extraction by Triton X-100. (Fig. 1B). In anattempt to circumvent this problem, different detergentswere tested for their ability to extract the membranes ofthe Toxoplasma pellicle as judged by electron mi-croscopy. Deoxycholate (DOC) in combination withcell homogenization was found to be the most efficientat removing all parasite membranes and routinelyyielded preparations in which all parasites were thor-oughly extracted as judged by electron microscopy ofthin sectioned and negatively stained samples (Fig.1C,D). Overall, the structure of DOC-extracted para-sites was found to be very similar to that of TritonX-100 extracted parasites. The previously described

Fig. 1. Extraction of Toxoplasma gondii with Triton X-100 andsodium deoxycholate. Isolated parasites were extracted with 1% Tri-ton X-100 (A, B) or 1% DOC (C, D) and analyzed by negativestaining (A, C) or thin sectioning (B, D). Arrowheads denote regionsof pellicular membranes not removed by Triton X-100 extraction.Extraction with DOC followed by negative staining (C) or thinsectioning (D). Bar=250 nm.


Fig. 2. Toxoplasma gondii possesses a cytoskeletal structure that envelops the entire organism. (A) Metal coating of whole mounts ofDOC-extracted parasites reveals that in addition to the microtubule-based cytoskeleton, Toxoplasma also possesses a new structure. This iscomposed of three distinct domains. The central domain of the subpellicular network is composed of interwoven filaments spaced about 30 nmapart (C). At the anterior end of the parasite, the structure appears to be composed of parallel arrangements of thicker filaments with diameterscomparable to microtubules (B). At the posterior end of the parasite (D), a smooth, cup-like is found. Bar=250 nm.

components of the microtubule-based cytoskeleton suchas the subpellicular and central microtubules, the polarrings and the conoid were clearly visible by electronmicroscopy of negatively stained deoxycholate-ex-tracted samples. It was noted, however, that the conoidappeared somewhat loosened in DOC-extracted cy-toskeletons when compared with those prepared usingTriton X-100. This may well be due to the apparentremoval of the anterior and posterior conoidal rings byDOC.

Further morphological characterization of the cy-toskeletal preparations was carried out by coating withpalladium. The polar ring and subpellicular micro-tubules were clearly visible in metal-coated preparations(Fig. 2A), but the conoid was visible only when itprotruded from the polar ring. In addition to thesestructures, we also observed a cytoskeletal structurethat had not been described in Toxoplasma thus far.This structure consists of a filamentous network thatextends from the polar ring to the extreme posteriorend of the parasite and envelops the subpellicular mi-crotubules (Fig. 2A). The individual filaments thatmake up the network are readily visible in the centralregion of the structure and have a diameter of 8–10 nm(Fig. 2C). At the extreme anterior and posterior ends ofthe parasite, the network is associated with distinctstructures. At the anterior end of the parasite (Fig. 2B),

the network is attached to parallel, 20–25 nm filaments,that may be subpellicular microtubules. At the poste-rior end of the parasite (Fig. 2D), the network appearsto be anchored to a smooth, cup-like structure with acentral opening. Overall, the network has the samegeneral shape as the intact parasite, suggesting that thisstructure may play a role in generating and maintainingcell shape. In addition, the exceptional resilience of thenetwork, as judged by its ability to survive the harshhomogenization methods employed, suggests it mayalso be a major source of mechanical strength for theparasite.

3.2. Immunological characterization of cytoskeletalpreparations

As a first step towards characterizing the composi-tion of the Toxoplasma cytoskeleton, we prepared poly-clonal antisera to a large-scale preparation ofDOC-extracted cytoskeletons. Reactivity of the anti-serum with different elements of the Toxoplasma cy-toskeleton was determined by indirectimmunofluorescence microscopy (Fig. 6A–C). Whereasmonoclonal antibodies to �-tubulin clearly react withthe conoid and subpellicular microtubules in DOC-ex-tracted parasites, the anti-CYT antiserum does notappear to react with these structures. Instead, this


Fig. 3. Deduced amino acid sequences of TgIMC1 (A) and TgIMC2(B). Sequences are available from GenBank under accession numberAY032678 for IMC1 and AY032682 for IMC2.

3.3. Molecular cloning of TgIMC1 and TgIMC2

An expression library of T. gondii cDNA in �ZAPwas screened with the anti-CYT antiserum and resultedin the identification of a number of reactive clones.Two of these contained open reading frames that en-coded novel proteins, which we named TgIMC1 andTgIMC2. Although no strong homologies to knownproteins were found, it was noted that weak homologiesexisted to the rod-like domains of a number of cy-toskeletal proteins, such as myosins and neurofilaments.Full-length cDNA clones were isolated for bothproteins. The TgIMC1 sequence predicts a protein of650 amino acids with a molecular weight of 70 kDa(Fig. 3A). The sequence is extremely rich in valine(13.3%) and glutamic acid (13.9%), which together rep-resent 27% of the total amino acid content. TgIMC1also contains cysteine-rich regions at its N and Cterminus. TgIMC2 is predicted to be a 1029 amino acidprotein with a molecular weight of 118 kDa (Fig. 3B).

Comparison of the deduced amino acid sequences ofboth proteins with known sequences contained in Gen-Bank and EMBL databases revealed only weak similar-ities to a number of cytoskeletal proteins, includingmyosins, trichohyalin, involucrin and other intermedi-ate filament proteins. We did note, however, that thesesimilarities were always confined to the domains ofthese proteins that are known to adopt a coiled-coilconfirmation. Based on this observation, we examinedthe predicted protein sequences of TgIMC1 andTgIMC2 for the presence of similar domains using theLupas algorithm [20]. Based on our analysis, bothTgIMC proteins are predicted to contain domains witha coiled-coil confirmation (Fig. 4B). In the case ofTgIMC1, the predicted coiled-coil region begins at

antiserum reacts with a structure that appears to en-velop the subpellicular microtubules and extends fromthe extreme anterior to the posterior end of the para-site. This pattern suggests that the proteins reactivewith this antiserum are part of the filamentous networkdescribed above.

Fig. 4. TgIMC1 and TgIMC2 are predicted to form coiled-coil structures. Diagram of the relevant homologies and predicted structural featuresin TgIMC 1 and TgIMC 2. Domain structure of TgIMC1 (A) and TgIMC2 (B) including the localization of the VPV repeat homologies (VPVrepeat), the predicted coiled coil (CC) region and the cysteine rich C-terminus (CRD). Sequence analysis of TgIMC1 (C) and TgIMC2 (D) usingthe COILS program. The probabilities of forming a coil coiled were calculated for each residue with the weighted MTIDK matrix using windowsof 14, 21 or 28-residue (shown).


Fig. 5. Expression of TgIMC1 and TgIMC2 in Toxoplasma tachy-zoites. Parasite extracts were analyzed for the expression of TgIMC1and TgIMC2 with monospecific antisera. The negative control lanewas incubated with only the secondary antibody. The anti-IMC1antiserum reacts with a protein with an apparent molecular weight of80 kDa. The anti-IMC2 antiserum reacts with a protein with anapparent molecular weight of 150 kDa. The 28 kDa protein (indi-cated by an asterisk) present in all lanes corresponds to immunoglob-ulin light chains present in Toxoplasma lysates prepared fromparasites grown in mice.

skeleton in a number of unicellular organisms. Betweenamino acids 50 and 450, TgIMC1 is 37% identical and45% similar to the articulins of Euglena gracilis. Thisappears largely due to the presence valine and proline-rich domains in both proteins.

3.4. TgIMC1 and TgIMC2 are localized to thesubpellicular network of Toxoplasma

For the further characterization of the TgIMCproteins, monospecific antisera were producedto recombinant proteins prepared in E. coli. As can beseen in Fig. 5, both TgIMC1 and TgIMC2 arereadily detected in Toxoplasma tachyzoites. TgIMC1antiserum reacts with a protein of approximately80 kDa. The TgIMC2 antiserum reacts with aprotein of approximately 150 kDa. Both TgIMC1 andTgIMC2 clearly migrate with a higher apparent molec-ular weight than predicted by the amino acid sequence.This may be a result of their high content of acidicamino acids or the presence of extended domains.Extensive post-translational modifications can probablybe ruled out as an explanation for the anomalousbehavior of these proteins during SDS-PAGE, as re-combinant TgIMC1 and 2 have the same apparentmolecular weights as their native counterparts (data notshown).

The monospecific antisera were used to localizeTgIMC1 and TgIMC2 in DOC-extracted parasitepreparations. As can be seen in Fig. 6, both proteinsare located in a structure that has maintained theoriginal shape of the parasite and that appears to

amino acid 223 and extends through amino acid 297.Sequence analysis of TgIMC2 predicts six regions ofcoiled-coils beginning at amino acid 253 and extendingthrough amino acid 772. All similarities to knowncytoskeletal proteins were confined these domains.

In addition to the similarities observed between theputative coiled-coil domain of TgIMC1 and mam-malian cytoskeletal proteins, we also observed similari-ties to the articulins, proteins that form the membrane

Fig. 6. TgIMC1 and TgIMC2 are present in the network. DOC-extracted parasites were double-labeled with antibodies to tubulin, and either theanti-CYT antiserum (A–C) or the monospecific antisera to TgIMC1 (D–F) and TgIMC2 (G–I).


Fig. 7. TgIMC1 and TgIMC2 are localized to the subpellicular network. DOC-extracted parasites were incubated with monospecific antisera toTgIMC1 (A, B), or no antiserum (C) followed by secondary antiserum conjugated to 10 nm gold. In DOC-extracted parasites, antisera to TgIMC1and TgIMC2 (data not shown) react with the network and no labeling of the microtubules is observed. Panel B is an enlargement of the boxedregion in panel A. Arrowheads mark gold particles associated with the network. For orientation, the microtubules are marked with white arrows.Bar=500 nm (A) and 100 nm (B,C).

surround the subpellicular microtubules. In order todetermine whether these proteins are components of thenetwork described above, we performed immunoelec-tron microscopy on whole specimens of DOC-extractedparasites. As can be seen in Fig. 7, antibodies toTgIMC1 react evenly with network filaments along theentire length of the parasite. Antibodies to TgIMC2give the same labeling pattern (data not shown), sug-gesting that both TgIMC1 and TgIMC2 are present inthe same network of filaments.

To localize the filamentous network in relationto the microtubule-based cytoskeleton and thedifferent pellicular membranes, we isolated pellicle/cy-toskeleton complexes and localized TgIMC1 andTgIMC2 in these structures by immunoelectron mi-croscopy. After a brief sonication of Toxoplasma, thepellicle and microtubule-based cytoskeleton remain rel-atively intact and can be separated from the cytoplas-mic material by differential centrifugation. Thesepreparations were incubated with TgIMC1 andTgIMC2-specific antisera and gold-conjugated sec-ondary antibodies, followed either by embedding andthe preparation of thin sections or by their absorptionto grids and metal coating.

In thin sections, TgIMC1 and TgIMC2-specific label-ing was present on the inner face of the pellicle betweenthe microtubules (Fig. 8). In whole mounts of labeledpellicles, the TgIMC1 and TgIMC2-specific label wasassociated with a filamentous network that lies immedi-ately underneath the inner membrane complex asshown in Fig. 8. These observations suggest thatTgIMC1 and TgIMC2 form a filamentous network thatis closely associated with inner face of the inner mem-brane complex and envelops the subpellicularmicrotubules.

3.5. P. falciparum contains an TgIMC1 homologue

Observations made previously in the apicomplexanparasites Sarcocystis and Besnoita, suggest that theseparasites also possess a subpellicular network. To deter-mine whether this structure is a common feature ofapicomplexan parasites, we searched available data-bases for sequence homologues of TgIMC1 and 2. Asingle homologue of TgIMC1 was detected in thegenome database of P. falciparum and was namedPfIMC1. The predicted amino acid sequences ofTgIMC1 and its homologue in Plasmodium demon-strate a high degree of sequence conservation, especiallyin the N and C-terminal domains as shown in Fig. 9A.When compared to TgIMC1, the predicted sequence ofPfIMC1 has an additional stretch of approximately 220amino acids near the C-terminus. This sequence con-tains seven repeats of the sequence N(E/N)N(S/I)N(G/N)KEI that do not match any known patterns. Thisregion may be an example of repeat sequences com-monly found in Plasmodium proteins [24]. On the otherhand, as the PfIMC1 sequence is derived from a ge-nomic sequence by removal of predicted introns, wecannot exclude the possibility that the apparent pres-ence of extra amino acids in this protein is due to thepresence of a non-recognized intron in the genomicsequence. Resolution of this issue awaits the isolationof the cDNA sequence encoding PfIMC1. Expressionand localization of PfIMC1 in P. falciparum was deter-mined by immunoblotting and immunofluorescence mi-croscopy. Using antisera to TgIMC1, a cross-reactiveprotein with the predicted molecular weight of PfIMC1is readily detected in P. falciparum-infected erythro-cytes (Fig. 9B). Immunofluorescence analysis with thesame antisera reveals only a weak and diffuse staining


pattern in all intracellular developmental stages of theparasite (data not shown), except in late schizonts andmerozoites. In these, the cross-reactive protein is clearlydetectable and localized to the periphery of the parasiteas shown in late schizonts in Fig. 9C. A P. falciparumhomologue of TgIMC2 has not been identified thus far.

4. Discussion

Here we report on the morphological and molecularcharacterization of the subpellicular network, a filamen-tous component of the membrane skeleton of the pro-tozoan parasite T. gondii. The subpellicular network isintimately associated with the cytoplasmic face of theToxoplasma pellicle and extends along its entire length.The majority of the network is composed of filamentswith an 8–10 nm diameter. At the anterior end of the

parasite, the network appears to be connected to thepolar ring by means of thicker filaments, which mayrepresent microtubules with associated proteins. At theposterior end of the parasite, the network appears to beanchored in a smooth, cup-like structure. We haveidentified two of the protein subunits of this structurein T. gondii, TgIMC1 and TgIMC2, as well as PfIMC1,a homologue of TgIMC1 in the malaria parasite P.falciparum. Both are novel proteins and only resembleknown animal cytoskeletal proteins through the pres-ence of putative coiled-coil domains. TgIMC1 is alsohomologous to the articulins, a family of proteins in-volved in the formation of the resilient membraneskeletons of E. gracilis and Pseudomicrothorax dubius.Like the membrane skeletons of these organisms, thesubpellicular network of T. gondii is very stable struc-ture that displays a remarkable mechanical strength. Itcan withstand the harsh cell homogenization proce-dures used in its isolation while largely maintaining theoriginal shape of the parasite. Based on these observa-tions, we propose that the subpellicular network is partof a membrane skeleton that is vital for the mainte-nance of cellular integrity and shape of T. gondii.

The organization of the different elements of theToxoplasma pellicle, the plasma membrane, inner mem-brane complex, the subpellicular network, and a micro-tubule-based cytoskeleton, is similar to that of thesurface complexes in a number of free-living protists. Ingeneral, these also consist of a plasma membrane,membrane cisternae, a membrane skeleton (or epi-plasm) and a microtubule-based cytoskeleton, althoughthe exact arrangement of these elements varies depend-ing on the organism. The main function of the mem-brane skeleton in these organisms is also believed to bethe maintenance of cell shape and mechanical stability[25].

Membrane skeletons consist, in general, of networksof filamentous proteins that are linked to a membranethrough interaction with integral membrane proteinsand are required for maintenance of cell integrity byproviding mechanical strength to the plasma mem-brane. In addition, they are involved in the organiza-tion of subcellular organelles, such as the animalnucleus. The importance of the membrane skeleton inthe maintenance of cellular and organellar integrity isillustrated by the effects of their disruption in a numberof hereditary disorders. Elliptocytosis and spherocyto-sis, caused by mutations in components of the erythro-cyte membrane skeleton, are accompanied by a markeddecrease in mechanical stability of these cells andhemolytic anemia [26]. Mutations in components of thenuclear lamina, as occurs in Emery-Dreifuss musculardystrophy, disrupt this structure and cause defects innuclear assembly and function [27].

The observation that the plasma membrane, innermembrane complex, the subpellicular network, and

Fig. 8. TgIMC1 and TgIMC2 are localized along the cytoplasmic faceof the inner membrane complex. Isolated pellicles were incubatedwith no antiserum (C), anti-IMC2 (B, D), and anti-IMC1 (A, E, F)and secondary antiserum conjugated with 10 nm gold. Samples werethen coated with metal (A,B) or embedded in plastic for thin section-ing (C–F). In intact pellicles, both antisera react with a networklocated on the cytoplasmic face of the inner membrane complex (A,B). In thin section experiments, labeling is only found on the inner,cytoplasmic face of the pellicle (D–F). Panel F offers an enlargedview of the boxed region in panel E and shows the presence of thesubpellicular microtubules (arrowheads) and the TgIMC-containingnetwork on the cytoplasmic face of the pellicle. Bar=250 nm (A, B)and Bar=125 nm (C–F).


Fig. 9. Plasmodium falciparum expresses a TgIMC1 homologue. (A) Protein sequence alignment of TgIMC1 and PfIMC1 (accession numberCAB38992.1). Sequences were aligned using ClustalW. Identical residues are shown with a black background, similar residues with a graybackground. The N(N/E)N(S/I)N(G/N)KEI repeats in PfIMC1 are underlined. (B) Lysates of uninfected (RBC) and P. falciparum-infected humanerythrocytes (Pf) were analyzed by immunoblotting, using a monospecific TgIMC1 antiserum. An arrowhead indicates the single reactive proteinin parasite-infected erythrocytes. (C) P. falciparum-infected erythrocytes were analyzed by immunofluorescence using a monospecific antiserum toTgIMC1. Nuclear staining with DAPI was used to determine the approximate developmental stage of the parasites. PfIMC1 appears to be locatedin the periphery of late stage schizonts.

subpellicular microtubules of Toxoplasma, remaintightly associated during the harsh procedures used toisolate the parasite pellicle, suggest that they interacttightly with each other. A direct association betweenthese two structures is likely, considering the closeapposition of the subpellicular network to the cytoplas-mic face of the inner membrane complex. The nature ofthis association is not known at this time, but couldinvolve the presence of lipid modifications on the net-work proteins, the interaction of network proteins withintegral membrane proteins in the inner membranecomplex, or a combination of the two.

At this time, there is no experimental evidence formodification of TgIMC1 and TgIMC2 by lipid addi-

tion. The N and C-terminal domains of both TgIMC1and PfIMC1 are rich in cysteine residues, which arecommonly used for covalent modification of proteinswith lipids, including prenylation, palmitoylation, andmyristoylation. Interestingly, TgIMC1 protein containsa consensus motif (CXC) for isoprenylation, a lipidmodification often involved in protein targeting tomembranes and essential for the correct targeting ofnuclear lamins A and B to the nucleus and theirefficient incorporation into the lamina [28,29].

In other protists with microtubule-based membraneskeletons like the Euglena, the organization of thesubpellicular microtubules has been linked to the pres-ence of regular arrays of intramembraneous particles


[13,30–34]. No integral membrane proteins of the in-ner membrane complex of Toxoplasma have beenidentified biochemically thus far but freeze-fractureanalysis of the pellicle has demonstrated the presenceof two populations of intramembranous particles(IMPs) in the inner membrane complex. One of these,consisting of double rows of IMPs, is believed to beassociated with the subpellicular microtubules basedon their distribution in the membrane. The secondpopulation of IMPs is found in single rows in theinner membrane complex [14–16]. Both IMP popula-tions are organized in the inner membrane complexwith a 32 nm periodicity that is maintained along theentire length of the parasite. It is unlikely that thesubpellicular microtubules play a role in the genera-tion or maintenance of this organization. Although themicrotubules extend only along the anterior two-thirdsof the parasite, the IMPs are organized along theentire length. Furthermore, disruption of the subpellic-ular microtubules does not result in an alteration ofthe IMP organization. It has therefore been proposedby Morrissette et al. [16] that an additional filamentsystem within the membrane skeleton of Toxoplasmais necessary to maintain both the uniform lateral spac-ing of the particles and their register from row to rowover long distances. The filaments of the subpellicularnetwork we describe here are organized in amanner that is similar to the distribution of the IMPsin the inner membrane complex, suggesting an associa-tion between the two structures. We propose that theIMPs are in fact integral membrane proteins that linkthe subpellicular network to the cytoplasmic face ofthe inner membrane complex, thus generating a re-silient membrane skeleton that imparts mechanicalstability on T. gondii and other apicomplexan para-sites.

Acknowledgements

We would like to thank Ed Philips, Eugene Armsand Julie Hopkins for technical assistance and DrNaomi Lang-Unnasch for providing asynchronous cul-tures of Plasmodium falciparum-infected erythrocytes.We are grateful to Dr Gail Johnson for providing amonoclonal antibody to �-tubulin. We would also liketo thank Drs Gary Ward, David Roos, David Sibleyand Ms Kim Carey for helpful discussions. Sequencedata for P. falciparum chromosome (1,3,4,5,6,7,8,9,13)was obtained from The Sanger Centre website at http://www.sanger.ac.uk/Projects/P– falciparum/. Sequencingof P. falciparum chromosome (1,3,4,5,6,7,8,9,13) wasaccomplished as part of the Malaria Genome Projectwith support by The Wellcome Trust. This researchwas funded in part by a New Investigator Award fromthe Burroughs Wellcome fund.

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