Identification and purification of actin from the subpellicular network of Toxoplasma gondii tachyzoites

International Journal for Parasitology 35 (2005) 883–894 www.elsevier.com/locate/ijpara

Identification and purification of actin from the subpellicular network of Toxoplasma gondii tachyzoites Araceli Patro´n Sa, Mo´nica Mondrago´na, Sirenia Gonza´lezb, Javier R. Ambrosioc, Alma L. Guerrero Bd, Ricardo Mondrago´na,* a

Departamento de Bioquı´mica, Centro de Investigacio´n y Estudios Avanzados del IPN. Av. Instituto Polite´cnico Nacional No 2508. Col. Sn Pedro Zacatenco, Del. Gustavo A. Madero., Me´xico D.F. C.P. 07360 b Unidad de Microscopia Electro´nica, Centro de Investigacio´n y Estudios Avanzados del IPN, DF/Me´xico c Departamento de Microbiologı´a y Parasitologı´a, Facultad de Medicina, Universidad Nacional Auto´noma de Me´xico, Me´xico d Departamento de Morfologı´a, Universidad Auto´noma de Aguascalientes Aguascalientes/Ags, Me`xico Received 17 January 2005; received in revised form 22 March 2005; accepted 31 March 2005

Abstract Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence of cortical actin has been widely suggested, visualisation and localisation of actin filaments has not been reported. The subpellicular cytoskeleton network is a recently described structure possibly involved in the dynamic events. Using non-ionic detergent extractions, the cortical cytoskeleton network was enriched and used for the isolation and identification of actin. Actin was detected by Western blots in extracts of cytoskeleton networks, and it was localised by gold staining in the network and in both the apical end and the posterior polar ring. Actin was isolated from subpellicular cytoskeleton extracts by binding to DNase I, and it polymerised in vitro as filaments that were gold-decorated by a monoclonal anti-actin antibody. Filaments bound the subfragment 1 of heavy meromyosin, although with atypical arrangements in comparison with the arrowheads observed in muscle actin filaments. Treatment with cytochalasin D and colchicine altered the structural organisation of the subpellicular network indicating the participation of actin filaments and microtubules in the maintenance of its structure. Actin filaments and microtubules, in the subpellicular network, participate reciprocally in the maintaining of the parasite’s shape and the gliding motility. q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Actin; Apicomplexans; Cytoskeleton; Colchine; Cytochalasin D; Motility; Subpellicular network; Toxoplasma gondii

1. Introduction Toxoplasma gondii is a highly motile obligate intracellular protozoan that invades cells through an active process dependent on actin-myosin interactions (Ryning and Remington, 1978; Schwartzman and Pfefferkorn, 1983; Hiroshi et al., 1995; Dobrowolski and Sibley, 1996; Dobrowolski et al., 1997a,b). Toxoplasma, like other apicomplexan parasites, lacks locomotion organelles, however, it displays highly dynamic twirling–gliding movements over the substratum, without changing its cell shape (King, 1988; Mondragon et al., 1994; Mondragon and * Corresponding author. Tel.: C52 55 5747 3800; fax: C52 55 5747 7083. E-mail address: [email protected] (R. Mondrago´n).

Frixione, 1996; Hakansson et al., 1999). Its cytoskeleton is constituted by a cage of 22 helical subpellicular microtubules organised from the polar ring and associated with the inner membrane complex (Dubremetz and Torpier, 1978; Russell and Sinden, 1981; Russell and Burns, 1984; Nichols and Chiappino, 1987). The conoid, a retractile apical organelle that is projected against the host cell plasma membrane during the active invasion (Chiappino et al., 1984; Werk, 1985), consists of spiral fibrous subunits (De Souza, 1974; Nichols and Chiappino, 1987; Morrissette et al., 1997) containing a tubulin (Hu et al., 2002). Tachyzoite motility and conoid extrusion are inhibited by cytochalasin D (CD), an actin filament disrupting drug as well as by butanedione monoxime (BME), a light chain ATPase inhibitor, suggesting the involvement of actin– myosin interactions (Dobrowolski and Sibley, 1996;

0020-7519/$30.00 q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2005.03.016

884

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

Mondragon and Frixione, 1996; Hakansson et al., 1999; Herm-Gotz et al., 2002). Although Toxoplasma is a highly dynamic parasite, the presence of actin filaments has been debated because of the difficulties detecting them with fluorescent fallacidin or phalloidin or with conventional ultrastructural EM methods (Cintra and De Souza, 1985; Bannister and Mitchell, 1995; Dobrowolski et al., 1997a,b; Poupel et al., 2000). In contrast, by immuno-gold and immunofluorescence methods, the actin molecule has been found at the conoid and associated with the plasma membrane (Cintra and De Souza, 1985; Endo et al., 1988; Yasuda et al., 1988; Allen et al., 1997; Dobrowolski et al., 1997a,b). Jasplakinolide, a membrane-permeable actinpolymerising and filament-stabilising drug (Bubb et al., 1994), induces the polymerisation of actin filaments at the apical end without the induction of the conoid extrusion and with a reversible inhibition of the cell invasion suggesting a role for the actin polymerisation–depolymerisation dynamics as a requisite for motility and invasion (Shaw and Tilney, 1999). Biochemical evidence indicates that actin is mainly found in tachyzoites as the globular monomer (G-actin) (Dobrowolski et al., 1997b; Poupel and Tardieux, 1999), because of the participation of two actin monomer-sequestering proteins, ADF (Allen et al., 1997) and toxofilin, which also caps actin filaments to induce their disassembly (Poupel et al., 2000). Detection of myosin molecule TgMyoA beneath the plasma membrane suggests its participation to provide the dynamic force for the parasite motility (Herm-Gotz et al., 2002). Recently, a complex of proteins that includes the actin binding protein aldolase was described to mediate the molecular interactions between the C domains of Plasmodium TRAP and Toxoplasma MIC2 and the actin cytoskeleton (Jewett and Sibley, 2003). Through extraction treatments, a cortical cytoskeleton composed of a subpellicular network associated with the subpellicular microtubules was reported, which was distributed along the tachyzoites (Mann and Beckers, 2001; Oliveira-Lima et al., 2001). TgIMC1 and TgIMC2, two proteins from the subpellicular network, are associated with the cytoplasmic face of the inner membrane complex between the subpellicular microtubules (Mann and Beckers, 2001). The subpellicular network assembles during the early stages of the development of daughter parasites, as a structure that gradually increases its stability associated with a proteolytic removal of the carboxy-terminus of TgIMC1 (Mann et al., 2002). High resolution SEM of detergent extracted tachyzoites revealed a net of actin-like filaments. The biochemical nature of this network had not been determined (Schatten et al., 2003). In the present study, actin was identified and isolated from the subpellicular network cytoskeleton. A function for actin in the cytoskeleton network was determined by the exposure to drugs that alter the cytoskeleton integrity. Location of actin at the subcortical level associated with the subpellicular network suggests its participation in the

dynamic behaviour of the parasite during gliding motility and cell invasion.

2. Materials and methods All reagents and protease inhibitors were purchased from SIGMA, Chemical Co (St Louis, MO), unless otherwise indicated. 2.1. Animals Balb/c mice used for parasite infections were maintained in an animal facility with regulated environment conditions of temperature, humidity and filtered air. Management was performed according to the country official norm NOM062-ZOO-1999 for the production, care and use of laboratory animals (Mexico). 2.2. Maintenance and purification of T. gondii tachyzoites RH strain tachyzoites were maintained by i.p. passages in female Balb/c mice (Mondragon et al., 1994). After cervical dislocation, parasites were recovered from i.p. exudates after a peritoneal washing with PBS (138 mM NaCl, 1.1 mM K2PO4, 0.1 mM Na2HPO4 and 2.7 mM KCl, pH 7.2) and purified by filtration through 5 mm pore polycarbonate membranes (Millipore Co, Bedford, MA). 2.3. Preparation of subpellicular networks for structural characterisation Tachyzoites (1!103/ml) were settled on 200 mesh nickel grids covered with formvar film (Polysciences, Warrington, PA) for 5 min. Parasites were rapidly washed with PHEM (10 mM HEPES, 10 mM EGTA, 1 mM MgCl2), 50 mg/ml N-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), 50 mg/ml Na-p-Tosyl-L-lysine choromethyl ketone (TLCK) and 17.4 mg/ml phenylmethylsulfonyl fluoride (PMSF) and incubated with 0.1% Triton X-100 in PHEM for 5 min, and then rinsed twice with PHEM to provide the subpellicular networks (modified from Hartwig, 1992). For structural analysis, networks were negatively stained with 1% uranyl acetate (Polysciences, Warrington, PA) and viewed through a JEOL 2000EX TEM at 80 keV (JEOL LTD, Japan). Other samples were dehydrated in increasing concentrations of ethanol, critical point dried in a CO2 atmosphere in a Samdryw-780A apparatus (Tousimis Research Co, USA) and coated by the evaporation of Platinum–carbon (Pt/C) rods at 458 in a BAF 400T freeze fracture system (Balzers, Austria) and then observed in the TEM. 2.4. Actin immuno-gold staining Subpellicular networks on the grids were blocked with 0.1% BSA in PHEM for 2 min and incubated overnight with

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

a Pan monoclonal C4 antibody against actin (C4 mAb) at 1:40 dilution (Chemicon International Inc, Temecula, CA) in 1% BSA-PHEM. The C4 mAb recognises all known actin isoforms including recombinant ACT1 from T. gondii (Dobrowolski et al., 1997b). Grids were thoroughly washed out with PHEM and then incubated for 3 h with goat antimouse polyclonal antibodies coupled to 20 nm colloidal gold particles (Zymed, San Francisco, CA) diluted 1:100 in PHEM. Grids were rinsed, fixed with 1% glutaraldehyde for 10 min and processed by critical point drying and Pt/C coating as was described above. Negative controls were samples incubated with a mAb against dystrophin IgG1 that shared the same isotype as the C4 mAb. 2.5. SDS-PAGE analysis of the networks The protein concentration of the extracts was determined by a modified Lowry’s method (Dunn, 1989). Samples were diluted with an equal volume of sample buffer (0.1 M Tris– HCl pH 6.8, 4% SDS, 20% glycerol and 0.2% bromophenol blue), and boiled for 5 min. Every lane was loaded with 20 mg of protein. Samples were separated by electrophoresis under reducing conditions in a 10% SDS-PAGE, according to Laemmli (1970). Broad range molecular weight standards (Bio-Rad laboratories Inc., Me´xico) were used. After electrophoresis, gels were silver stained or transferred to nitrocellulose sheets for Western blot. Actin purified from rabbit muscles according to Pardee and Spudich (1982) was used as a positive control in all the assays. 2.6. Silver staining Gels were stained following the rapid silver staining method (Coligan et al., 1995). Briefly, gels were washed out with milli-Q purified water, immersed in fixative solution (0.018% formaldehyde in 40% methanol) for 10 min, submerged in 0.02% sodium thiosulfate for 1 min, washed out again and incubated with 0.1% silver nitrate solution for 10 min. Developing was performed by incubation with thiosulfate developing solution until a brown precipitate was formed. The reaction was stopped with a solution containing 50% methanol and 12% acetic acid. 2.7. Actin identification in the subpellicular networks by Western blot analysis Polyacrylamide gels were transferred for 2 h to 45 mm pore nitrocellulose membranes (Bio-Rad Laboratories Inc, Me´xico) in a HEP-1 semi-dry transfer system (Owl Separation Systems, Portsmouth, NH). After transference, nitrocellulose membranes were immersed in the blocking solution 1 (Roche Diagnostics Co., Indianapolis, IN) diluted with TBS (10 mM Tris–HCl and 75 mM NaCl, pH 8.0) overnight at 4 8C and then cut in strips. The C4 mAb was diluted in the blocking solution 2 (Roche Diagnostics Co., Indianapolis, IN) in TBS and incubated with the strips for

885

2 h at room temperature. Strips were rinsed in TBS-T (1% Tween 20 in TBS) for 5 min and incubated with a polyclonal goat anti-mouse IgG conjugated to HRP (Zymed Lab., San Francisco, CA) diluted 1:4000 in the blocking solution 2 for 2 h at room temperature. Strips were thoroughly washed out and the antibody complexes were revealed by chemioluminescence with the enzyme substrate kit ECLe Western blotting analysis system (Amersham Biosciences, UK). 2.8. Isolation and solubilisation of the subpellicular networks for biochemical analysis Tachyzoites (6!107 cells) were treated with 0.1% Triton X-100 in PHEM for 5 min in order to obtain the subpellicular networks. Networks were sedimented by centrifugation at 150,000!g for 15 min and the pellet was washed out with PHEM by centrifugation. Pellets were solubilised by agitation with 1% Triton X-100 in buffer A (2 mM Tris–HCl, pH 8, 0.2 mM Na2ATP, 0.5 mM b mercaptoethanol, 0.2 mM CaCl2 and 0.005% NaN3) for 2 h. Solubilised components were precipitated by addition of 5 vol. of cold acetone. After centrifugation, pellets were solubilised in buffer A. 2.9. Actin purification by affinity column of DNase I Ten mg of DNase I (Sigma Co, St Louis, MO) were coupled to CNBr activated-sepharose-4B according to the Etoh’s method (Etoh et al., 1990). For actin isolation, solubilised subpellicular networks from 6!107 tachyzoites in buffer A, or actin purified from rabbit muscle according to Pardee and Spudich (1982) method were loaded into the DNase I column and incubated overnight at 4 8C and then thoroughly washed out with 0.1 M KCl in buffer A. Elution of the bound actin was performed by the sequential addition of 0.5% sodium deoxycholate (NaDOC) in 20 mM Tris– HCl, pH 8.3; 2% NaDOC in 50 mM glycine, pH 9.3 and 2% NaDOC in 50 mM diethylamine, pH 11.5. Fractions were analysed in a DU 650 spectrophotometer (Beckman, Fullerton, CA) at 280 nm, and those fractions corresponding to the elution peak were thoroughly dialysed against buffer A for 24 h under continuous agitation. An aliquot of the dialysed protein was precipitated with 5 vol. of acetone and about 20 mg were tested by Western blot for the presence of actin with the C4 mAb. 2.10. Actin polymerisation, immuno-gold staining and observation by TEM Aliquots of 10–50 mg of the actin rich fraction eluted from the DNase I column were incubated under actin polymerisation conditions in buffer B (50 mM KCl, 2 mM MgCl2 and 1 mM Na2ATP). Filaments were centrifuged at 150,000!g for 2 h, suspended in 20 ml of fresh buffer B and deposited on formvar coated grids. Samples were

886

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

stained with 2% uranyl acetate and observed in the TEM at 80 keV. For actin immuno-gold staining, actin filaments deposited on the grids were incubated overnight with the C4 mAb (Dil 1:40) in buffer B. After washings with buffer B, grids were floated on goat anti-mouse polyclonal antibodies coupled to 20 nm colloidal gold particles and negatively stained with 2% uranyl acetate and viewed through the TEM. 2.11. Actin filament decoration with myosin subfragment 1 Actin filaments polymerised in buffer B were incubated with heavy meromyosin subfragment 1 (S1, isolated from rabbit, SIGMA Chemical Co., St Louis, MO) according to Moore et al. (1970). Briefly, an aliquot of the polymerised filaments were deposited on formvar covered grids for 30 min. Grids were washed out with 0.6 M KCl and incubated for 15 s with 0.2 mg/ml of S1. Grids were rinsed with 0.2 mg/ml cytochrome c in 0.1% amylic alcohol at 4 8C. Filaments decorated with S1 subfragment were negatively stained with uranyl acetate and observed in the TEM. Formation of actin filaments: S1 subfragment complexes was confirmed by the presence of the typical arrowheads arranged along the filaments. 2.12. Treatment of the subpellicular networks with CD and colchicine Grids containing isolated subpellicular networks were exposed to 1–20 mM CD in PHEM or to 10 and 20 mM colchicine in PHEM. Controls for CD included the exposure to the respective concentration of the solvent dimethylsulfoxide (DMSO), while for colchicine, the controls were cytoskeletons maintained in PHEM during the period of the experiment. Samples were exposed to both drugs for 0.5, 1 and 2 h at room temperature. After incubation, cytoskeletons were fixed for 30 min with 1% glutaraldehyde and then critical point dried, Pt/C coated and observed in the TEM.

3. Results 3.1. Isolation of subpellicular networks Mild extractions of the tachyzoites (Fig. 1A) with the non-ionic detergent Triton X-100 allowed the reproducible isolation of the subpellicular cytoskeleton (Fig. 1B), consisting of the conoid, polar rings and 22 subpellicular microtubules associated with a network of longitudinal filaments parallel to the subpellicular microtubules and heavily cross-linked by numerous short fibres and extended along the entire length of the parasite. Although detergent treatment released cytoplasm organelles such as dense granules, rhoptries, micronemes, nuclei, etc., the typical crescent shape of the tachyzoites was preserved. 3.2. Distribution of actin in the subpellicular network Actin was detected by immuno-gold labeling with the C4 mAb distributed throughout the subpellicular network but not over the subpellicular microtubules confirming the specificity of the antibody and the method used (Fig. 2A, black arrowhead). Apparently, the diffuse appearance of the network after the immuno-gold decorations was due to the extent of the incubation periods and the multiple washings involved (Fig. 2A). Gold labeling was found decorating network remnants associated with the apical polar ring (Fig. 2A, apr and black arrows), suggesting its function as an anchorage organelle for the network at the apical end. Immuno-gold decoration was also observed associated with the conoid (c). Some posterior rings were found detached from the network (Fig. 2B), showing their fine structure constituted by two concentric rings. The inner ring is connected with the outer through short fibrous material

2.13. Release of actin monomers from the subpellicular network by CD treatment Subpellicular networks isolated from 5!107 tachyzoites, were incubated with 5 mM CD in PHEM with protease inhibitors during 0.5, 1 and 2 h at room temperature. Samples were centrifuged at 150,000!g for 10 min at 4 8C and the supernatant was recovered and precipitated with an equal volume of isopropanol overnight at 4 8C. Samples were centrifuged again and the precipitates were analysed by SDS-PAGE and Western blot with the C4 mAb.

Fig. 1. Subpellicular network of Toxoplasma gondii tachyzoite. (A) Longitudinal section of an intact tachyzoite; (B) Subpellicular network obtained after a mild extraction with Triton X-100 in PHEM solution, shows the microtubule organising centre (MTOC), the 22 subpellicular microtubules and the subpellicular network consisting of crosslinked filaments extending throughout the parasite. The posterior end is amplified in the inset. C, conoid; DG, dense granules; Mt, subpellicular microtubules; N, nuclei; Nt, subpellicular network; ppr, posterior polar ring. BarsZ 500 nm.

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

Fig. 2. Distribution of actin in the subpellicular network. (A) Actin immuno-gold detection with the C4 mAb was found distributed throughout the subpellicular network (arrowhead) but not over the subpellicular microtubules; (B) Correspond to a released posterior polar ring with remnants of the network and immuno-gold decorated with the C4 mAb (arrows). The arrowhead indicates interconnecting fibres between the two posterior rings; (C) Corresponds to the negative control of the subpellicular network incubated with an irrelevant monoclonal antibody against dystrophin molecule. apr, apical polar ring; c, conoid; Mt, microtubules; Nt, subpellicular network. BarsZ200 nm.

887

Fig. 3. SDS-PAGE and Western blot of subpellicular networks. (A) SDSPAGE and silver staining of: lane 1, molecular weight standards; lane 2, whole extract of tachyzoites; lane 3, extract of isolated subpellicular networks; lane 4, soluble fraction of the subpellicular network isolation; lane 5, control of rabbit muscle actin; (B) Western blot anti actin with the C4 mAb in: lane 1, subpellicular networks; lane 2, rabbit muscle actin.

corroborating the immune-gold detection on the subpellicular networks. (arrowhead in Fig. 2B). Only the outer ring was associated with the network at the posterior end showing higher actin labeling than the rest of the network suggesting a different concentration of actin in this zone (Fig. 2B, arrows). The inner posterior ring was also immuno-gold actin labeled. Filaments from the network were associated with the outer ring of the posterior pole following a spiral arrangement all over the tachyzoite towards the apical end. Controls of cytoskeleton samples incubated with an irrelevant antibody against dystrophin (with the same isotype of C4 mAb) did not show any specific labeling (Fig. 2C). 3.3. Electrophoretic and Western blot analysis of the subpellicular networks Extracts from tachyzoites and subpellicular networks were analysed by Western blot in order to determine the presence of actin (Fig. 3). Fig. 3A shows the electrophoretic pattern of a tachyzoite whole extract (lane 2), the insoluble fraction after the Triton X-100 treatment corresponding to the subpellicular network (lane 3), the soluble fraction after the Triton X-100 treatment (lane 4) and a rabbit muscle actin fraction purified according to Pardee and Spudich (1982) (lane 5). At least 28 bands were detected in the subpellicular network extracts distributed in a broad range of molecular mass between 30 and 200 kDa (lane 3). Actin was detected by Western blot as a 45 kDa band by the C4 mAb in the subpellicular network fraction (Fig. 3B),

3.4. Isolation of actin from rabbit muscle and solubilised subpellicular networks by DNase I affinity chromatography Actin purification from rabbit muscle and subpellicular network, was based on the specific binding property of the enzyme DNAse I, to IB and IIB actin subdomains (Sheterline et al., 1998). Muscle actin purification by DNase I chromatography showed a maximal actin peak after the elution buffer of pH 9.3 (Fig. 4A(a)). The SDSPAGE analysis of the eluted fraction showed a unique band with a molecular weight of 45 kDa (Fig. 4A(b), lane 2) which was subsequently identified as actin by Western blot with the C4 mAb (Fig. 4A(b), lane 3). Fractions of subpellicular networks, previously solubilised in buffer A, were applied to the DNase I column (Fig. 4B(a)) and treated with exactly the same series of elution buffers as was done for muscle samples. Similar to the binding observed with muscle actin, actin from the subpellicular network of T. gondii was purified by the DNase I method, although its elution occurred at pH 8.3 in contrast to muscle actin which eluted at pH 9.3. These differences could indicate the presence of slight structural variations between both actins that affect their binding to the DNase I molecule. The SDS-PAGE analysis of this fraction showed an enriched band with a molecular mass of about 45 kDa (Fig. 4B(b), lane 2) which was recognised in the Western blot as actin by the C4 mAb (Fig. 4B(b), lane 3).

888

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

Fig. 4. Isolation of actin from rabbit muscle and solubilised subpellicular networks by DNase I affinity chromatography. A(a), Graph of muscle actin fractionation by a DNase I affinity column after elution at pH 8.3, 9.3 and 11.5. A(b), SDS-PAGE and silver staining of: lane 1, whole extract of rabbit muscle actin loaded into the DNase I column; lane 2, eluted fraction peak at pH 9.3; lane 3, Western blot with the C4 mAb of the eluted fraction at pH 9.3. B(a), Graph of subpellicular network actin fractionation by DNase I affinity column after elution at pH 8.3, 9.3 and 11.5. B(b), SDS-PAGE and silver staining of: lane 1, solubilised subpellicular networks loaded into the column; lane 2, protein eluted from DNase I column at pH 8.3; lane 3, Western blot with C4 mAb in extracts of the eluted protein from the DNase I column at pH 8.3.

3.5. Polymerisation of actin purified by DNase I affinity chromatography In order to determine the in vitro polymerisation properties, actin aliquots isolated from the subpellicular network and rabbit muscle were incubated under polymerising conditions in buffer B, and then negatively stained and viewed through the TEM. In both cases, numerous filaments polymerised with similar appearances (Fig. 5A,B, black arrows). In the case of polymerised actin from the subpellicular network, certain annular structures of unknown nature were occasionally associated with the bundles of filaments (Fig. 5B, arrowhead).

muscle actin (Fig. 6C) or subpellicular network actin (Fig. 6D), indicating that these filaments contain actin. Interestingly, in the polymerised preparations from the subpellicular network actin, several structures presenting

3.6. Immuno-gold identification of polymerised filaments from isolated actins Filament bundles of in vitro polymerised actin, purified from muscle or subpellicular networks by affinity to the DNase I, were immuno-gold labeled by the C4 mAb. Negative controls included polymerised filaments from muscle actin (Fig. 6A) and from the subpellicular network actin (Fig. 6B) incubated with a IgG1 mAb against dystrophin and then with the secondary antibody coupled to the gold label. Both samples were negative for immuno-gold decoration. The C4 mAb specifically and abundantly labels polymerised filaments from purified

Fig. 5. Actin filament polymerisation in vitro. Negative staining electron micrographs of the fractions eluted from the column of DNase I and then incubated under polymerising conditions. (A) Muscle actin fraction eluted at pH 9.3; (B) Subpellicular network fraction of T. gondii eluted at pH 8.3. Black arrows indicate the filaments while the arrow head in B indicates the presence of annealed structures. BarsZ100 nm.

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

889

3.8. Effect of cytoskeleton depolymerising drugs on the subpellicular network structure

Fig. 6. Actin immuno-gold staining and S1 decoration of actin filaments purified by the DNase I column. (A) Negative staining micrographs of muscle actin filaments (negative control) incubated with a primary antibody against dystrophin IgG1 and a secondary antibody conjugated to gold particles; (B) Negative control of the actin filaments purified from the subpellicular network and incubated as in A; (C) Immuno-gold decoration of muscle actin incubated with primary C4 mAb (arrows); (D) Immunogold decoration of actin filaments purified from the subpellicular network with C4 mAb (arrows); (E) Striated structures found in the preparations of actin filaments from the subpellicular networks; (F) Muscle actin filaments decorated by S1 fragment (arrow), symbol (C) indicates the growing end of the filament; (G) Filaments of actin from the subpellicular network interacted with S1 fragment. The arrow indicates the S1 associated to the filament; the arrowhead shows an annealed structure associated with the filament. BarsZ100 nm.

In order to determine the participation of actin filaments and microtubules in the structural organisation of the subpellicular network, cytoskeletons were treated with CD, an actin filament depolymerising drug, as well as with colchicine, a microtubule depolymerising drug. For the different concentrations of CD used, controls were done with the equivalent concentration of the solvent DMSO. Treatment with 5 mM CD produced a gradual alteration of the subpellicular network integrity. After 30 min of exposure, the longitudinal filaments and their cross-linking fibres became fuzzy without a clear resolution under the TEM (Fig. 7A). At 1 h of exposure, the network became drastically altered at the middle of the tachyzoite, in the region of the cytoskeleton lacking subpellicular microtubules (Fig. 7B, arrowhead). Zones of the network which are spatially associated with the subpellicular microtubules or with the posterior ring were more preserved. However, after 2 h of treatment, the posterior pole containing the network was completely released leaving the anterior ends with the subpellicular microtubules and remnants of the network (Fig. 7C). The helical arrangement of the microtubules was completely disturbed after the release of the network. Treatment with 1 mM CD causes the appearance of cartwheel-like arrangements of the subpellicular microtubules freed from the subpellicular network (Fig. 7D). The conoid and polar ring were located in the centre of this structure. Two hours of exposure to 20 mM CD caused a complete disorganisation of the network and only

repetitive arrangements were observed although they had a faint labeling with the C4 mAb (Fig. 6E). Similar structures were previously reported as crystals in pure preparations of light meromyosin formed after a fall in the ionic strength (Huxley, 1963). Annular structures previously mentioned were not labeled by the antibody (data not shown). 3.7. Decoration of filaments with heavy mero-myosin subfragment 1 S1 myosin subfragment decoration is a standard assay that establishes the biochemical identity as well as the structural polarity of actin filaments. Filaments from muscle actin formed the typical arrowhead alignments with S1 decoration (Fig. 6F). In the case of polymerised filaments from network actin, a repetitive arrangement of S1 was observed distributed along the filaments (Fig. 6G, black arrow), although typical arrowheads were not observed, suggesting differences in the interaction of Toxoplasma actin and S1. Annular structures were additionally observed associated with the filaments (arrowhead).

Fig. 7. Effect of cytochalasin D (CD) on the subpellicular network integrity. Electron micrographs of subpellicular networks exposed to 5 mM CD for: (A) 0.5 h; (B) 1 h; (C) 2 h; (D) Distribution of subpellicular microtubules associated with the anterior polar ring and the conoid after treatment with 1 mM CD for 1 h; (E) Posterior polar ring after treatment with 20 mM CD for 2 h; (F) Remnant of an anterior polar ring associated with fragmented microtubules after treatment with 20 mM CD for 2 h. apr, anterior polar ring; ppr, posterior polar ring; Mt, microtubules; Nt, subpellicular network. BarsZ100 nm.

890

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

Fig. 8. Release of actin molecules from the subpellicular network by the treatment with CD; (A) SDS-PAGE and silver staining of the soluble fraction recovered after exposure of subpellicular networks to 5 mM CD. Lane 1 corresponds to the drug control with 0.01% DMSO for 2 h; Network was exposed to CD for: lane 2, 0.5 h; lane 3, 1 h; lane 4, 2 h. Arrows indicate the component released after the CD treatment. (B) Western blot of subpellicular network supernatants with the C4 mAb after exposure to 5 mM CD. Lane 1 corresponds to the control without CD and with 0.01% DMSO; lane 2, 0.5 h of CD exposure; lane 3, 1 h of CD exposure; lane 4, 2 h of CD exposure.

remnants of the posterior ends remained (Fig. 7E). Anterior rings still associated with damaged conoids were observed with isolated microtubules (Fig. 7F). The depolymerisation effect of CD on the actin filaments contained in the subpellicular network was additionally demonstrated by the release of actin molecules into the supernatant after the treatment with 5 mM CD during 30, 60 and 120 min (Fig. 8). The SDS-PAGE separation and silver staining of the respective supernatants showed a band of 45 kDa that was evident after 60 and 120 min of drug treatment (Fig. 8, lanes 3 and 4, arrow). Shorter times of exposure (30 min) did not show any band (Fig. 8, lane 2). Negative control of networks incubated only with DMSO did not produce the release of actin. In all the cytoskeleton samples exposed to CD were detected several bands of high molecular masses suggesting these would be components associated with actin filaments in the subpellicular network and released after the effect of the CD (data not shown). The 45 kDa molecule released by CD treatments was identified as actin by Western blot analysis using the C4 mAb (Fig. 8B lanes 3 and 4). In order to determine the role of the subpellicular microtubules in maintaining the structural arrangement of the subpellicular network, cytoskeleton preparations were

Fig. 9. Effect of colchicine on the subpellicular network structure. Electron micrographs of subpellicular networks exposed to 10 mM colchicine for: (A) 0.5 h; (B) 1 h; (C) 2 h. There was a modification in the organisation of the microtubules as well as the separation of the subpellicular network from the cage of the subpellicular microtubules. apr, anterior polar ring; Mt, microtubules; Nt, subpellicular network; ppr, posterior polar ring. Bars Z100 nm.

exposed to colchicine. Treatment of the subpellicular networks with 10 mM colchicine for 30 min produced a progressive detachment of the subpellicular network from the apical end (Fig. 9A), leaving the cage of subpellicular microtubules as a free structure (Fig. 9B). The structural organisation of the network was not modified by the drug treatment but the subpellicular microtubules modified their helical arrangements and integrity (Fig. 9B). At 2 h of treatment, isolated subpellicular networks were found completely released from the microtubular cage but preserving the posterior polar ring (Fig. 9C). These results indicate that the stability of the subpellicular cytoskeleton is maintained by the integrity of both subpellicular microtubules and actin filaments.

4. Discussion T. gondii uses actin–myosin interactions to power processes such as gliding–twirling motility, conoid extrusion and host cell invasion (Dobrowolski and Sibley, 1996; Dobrowolski et al., 1997a). These very fast dynamic processes occur in a few seconds and the existence of preformed actin filaments in Toxoplasma has been the subject of controversy, because conventional visualisation methods such as fluorescence and EM fail to identify microfilaments (Cintra and De Souza, 1985; Bannister and Mitchel, 1995; Dobrowolski et al., 1997a,b; Poupel et al., 2000; Oliveira et al., 2001). The identification of two T. gondii actin monomer-sequestering proteins, ADF and toxofilin suggests that actin is mainly kept in the cytoplasm as the monomeric form (Allen et al., 1997; Poupel et al., 2000). The recently described subpellicular network (Oliveira et al., 2001; Mann and Beckers, 2001) represents a structural candidate to provide the required molecular interactions responsible for the tachyzoite motility. Using mild extractions with Triton X-100, the subpellicular network was reproducibly isolated as a cross-linked network associated with most of the apical complex organelles, including subpellicular microtubules, conoid, anterior polar ring and posterior polar ring (Fig. 1B). TgIMC1 and TgIMC2 are two molecules recently described as part of the subpellicular network that are located at the cytoplasmic face of the inner membrane complex (Mann and Beckers, 2001). In an additional report, Oliveira et al. (2001) proposed that the subpellicular network is located beneath the plasma membrane and is closely associated with the inner membrane complex, suggesting the presence of actin in this structure. In a recent report, a molecular complex containing aldolase was described in T. gondii as a bridging molecule between actin cytoskeleton and adhesin molecules of the thrombospondin-related anonymous protein family (TRAP) such as MIC2 (Jewett and Sibley, 2003). Although aldolase complex has been suggested to participate in parasite motility, its localisation has been mainly detected at the apical end, probably participating in stages of gliding and cell invasion wherein the apical end is

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

involved. In addition, the short actin filaments found distributed throughout the subpellicular network would function to provide the continuity of the gliding motility functioning throughout the parasite pellicle. Evidence for the presence of actin in the subpellicular network was demonstrated by immuno-gold labeling with the C4 monoclonal antibody which recognises all the known actin isoforms (Lessard, 1988) including recombinant ACT1 from T. gondii (Dobrowolski et al., 1997b) (Fig. 2). Actin was distributed throughout the subpellicular network and associated with the conoid. The posterior polar ring showed heavy staining, suggesting a large amount of actin at the posterior end of the network (Fig. 2). Two types of filaments have been proposed to form the network, 10 nm diameter filaments proposed as intermediate filaments and actin like filaments of 7 nm (Schatten et al., 2003). The immuno-gold labeling for actin was found associated with the network, but not over the filaments, suggesting these filaments do not contain actin. We predict that actin would be present as short filaments distributed throughout the subpellicular network probably interacting with myosin molecules cortically distributed to provide the mechanical force for gliding motility (Meissner et al., 2002). Interestingly, the anterior and posterior polar rings delimit the network ends and actin is present in both structures. A similar distribution had been seen using immunofluorescence (IF) labeling, although it was not possible to determine the precise location and the structures that actin resides in (Cintra and De Souza, 1985; Endo et al., 1988; Dobrowolski et al., 1997a; Opitz and Soldati, 2002). Using immuno-gold and IF assays with the C4 mAb, actin molecules were found in the inner membrane complex (IMC), while myosin, detected by specific antibodies, was found associated with the plasma membrane in glycerol swollen tachyzoites (Dobrowolski et al., 1997a). Actin association with the anterior and posterior rings suggests the existence in these structures of actin binding proteins that would be maintaining this subcellular distribution. The function of posterior polar ring is still unknown, however, it was possible to characterise its fine structure as a double ring structure interlinked by fibrous branches of unknown composition (Fig. 2B). Existence of actin in the posterior polar ring suggests an interaction with myosin which is a resident component of this zone (Schwartzman and Pfefferkorn, 1983) to induce the clockwise rotation triggered after substrate adhesion and the twirling displayed during gliding or cell invasion (Mondrago´n et al., 1994; Hakansson et al., 1999). Presence of actin as a 45 kDa molecule was additionally corroborated in the subpellicular networks by Western blot with the C4 mAb (Fig. 3B, lane 1), an antibody that had been used to detect actin in whole tachyzoite extracts or the ACT1-GST fusion protein in Escherichia coli (Dobrowolski et al., 1997b). The electrophoretic SDS-PAGE pattern of subpellicular network extracts, showed at least 28 proteins (Fig. 3A, line 3), probably corresponding to components associated with the

891

subpellicular network including conoid, polar rings, subpellicular microtubules and the inner membrane complex, most of them of unknown nature. Actin was purified from the subpellicular network by DNase I affinity chromatography, a method successfully used for actin purification from different muscle and nonmuscle sources including actin related proteins from the bacteria Anabaena spp. and E. coli and it is based in a specific binding with actin IB and IIB domains (Etoh et al., 1990; Guerrero et al., 1996; Sheterline et al., 1998). Several conventional methods were tested to purify actin from the subpellicular network, including polymerisation–depolymerisation although with very low efficiencies (data not shown). The reported actin elution procedure for the DNAse I column using changes in ionic strength in the presence of mild denaturing treatments with 40% formamide or 3 M guanidium hydrochloride (Lazarides and Lindberg, 1974; Etoh et al., 1990; Zechel, 1984) produced a low actin recovery from the subpellicular network samples (data not shown). In contrast, elution by changing the pH resulted in a reproducible and efficient isolation of actin monomers from both muscle and subpellicular networks which were then studied by Western blot assay, in vitro polymerisation, immuno-gold labeling with the C4 mAb and S1 decoration. Differences in the elution pH for subpellicular network actin and muscle actin could be related to spatial differences that determine variations in the affinity for DNAse I molecule considering that only folded actin binds DNAse I with a very high affinity (O10K9 M) (Thulasiraman et al., 2000). More structural studies are necessary to solve this question. To our knowledge, this is the first report of actin isolation from T. gondii subpellicular networks. Successful isolation of actin monomers by binding to DNase I also indicates the presence of IB and IIB domains in the actin molecule. Although ACT1 of T. gondii differs from related actins at amino acids 269 to 281, 223 to 225 and 27 to 46, which occur at the external surface of the molecule (Dobrowolski et al., 1997b; Kabsch et al., 1990), these do not affect the binding of T. gondii actin monomers to DNase I as was additionally shown by immunofluorescence (Dobrowolski et al., 1997b) or by an actin inhibition binding assay in Plasmodium falciparum (Field et al., 1993). Interestingly, certain annular and striated structures similar to myosin crystals previously reported by Huxley (1963), were observed in the purified actin from the subpellicular networks, probably formed by co-purified actin associated proteins (Figs. 5B and 6E). Binding to heavy meromyosin fragment 1 (HMM1) or subfragment 1 (S1) of myosin is a functional assay for actin filaments. Both HMM1 and S1 are myosin proteolytic fragments that preserve the globular head of myosin that interacts with actin filaments (Carlier et al., 1994; Sheterline et al., 1998). HMM1 or S1 interaction with actin filaments generates an arrowhead pattern that indicates the filament polarity, where the barbed end represents the growing end of the filament (C) (Moore et al., 1970). Actin filaments purified from

892

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

the subpellicular network were decorated with the S1, although they have an asymmetrical arrangement (Fig. 6G) in comparison with the arrow head alignment pattern observed along muscle actin filaments (Fig. 6F). These differences may be due to previously reported variations in the actin amino acid sequences in T. gondii (Dobrowolski et al., 1997b). Changing the two or three amino-terminal acidic residues of chicken muscle b-actin reduced or modified the S1 binding pattern but not the function (Aspenstrom and Karlsson, 1991). Although the muscle actin amino acid sequence involved in the S1 binding is located in a divergent region of Toxoplasma actin, it does not eliminate the function of this region during motility. A complementary structural role for microtubules and actin filaments in the maintenance of the subpellicular network structure was seen after the treatment with CD and colchicine. Treatment with CD produced a disorganisation of the subpellicular network with the consequent release of actin monomers into the supernatant (Figs. 7 and 8), suggesting the presence of pre-formed actin filaments and their importance in the network stability. Although microtubule integrity was not affected by the CD, their subpellicular helical arrangement was completely lost and modified into a cartwheel-like organisation, with the subpellicular microtubules anchored to the centralised polar ring and conoid (Fig. 7D). A reciprocal structural stability of the network was also determined for the subpellicular microtubules using colchicine which is a drug that inhibits tubulin polymerisation (Tsuyama and Takenaka, 1997). After colchicine treatment, the subpellicular cytoskeleton suffered a severe structural modification with ruptures and release of the subpellicular networks which were found as isolated intact structures (Fig. 9). These findings suggest that actin filaments and microtubules have a complementary structural role in stabilising the subpellicular scaffold cytoskeleton in order to maintain the cell shape. Although filaments and microtubules are not in direct contact in the pellicle, their dispositions suggest the presence of unknown anchorage molecules between the subpellicular network and the subpellicular microtubules. Gephyrin has been described as a molecular linkage between microtubules and actin filaments in cortical microfilament webs of brain cells with participation of actin-associated proteins such as profilin and Mena/VASP (Giesemann et al., 2003). According to the finding of short actin filaments associated with the subpellicular network, we propose a motility model that considers two different possibilities (Fig. 10). During the induction of motility, myosin molecules located at the pellicle as was previously suggested (Allen et al., 1997; Opitz and Soldati, 2002) and at the plasma membrane (Dobrowolski et al., 1997a) would interact with the short actin filaments anchored to the subpellicular network (Fig. 10(I)). Anchorage of myosin molecules to the plasma membrane would be through unknown components with similar functions to those

Fig. 10. Toxoplasma gondii motility model based on the association of short actin filaments with the subpellicular network. Activation of Toxoplasma motility would involve two possibilities. (I) after adhesion to the subtrate by MIC2, MyoA molecules located at the pellicle or plasma membrane interact with short actin filaments anchored to the subpellicular network to display motility. MyoA may be associated to the cytosolic face of the plasma membrane through bridging molecules of unknown nature (MyoA docking protein, MADP). The second possibility (II) considers the anchorage of MyoA located at the IMC to the short actin filaments at the subpellicular network. This molecular arrangement would become joined to the plasma membrane through the aldolase complex according to Jewett and Sibley (2003). Because myosin–actin interactions take place on structures distributed in counter-clockwise arrangements such as the inner membrane complex, the subpellicular network and the subpellicular microtubules, the tachyzoites display gliding movements with twirls in a clockwise sense as also occur during cell penetration.

suggested for the MyoA docking protein (MADP) proposed in the glideosome model (Opitz and Soldati, 2002). These molecular interactions would produce a forward movement of the parasite while the plasma membrane becomes displaced in the opposite direction forward to the posterior end in a capping like process (Dobrowolski et al., 1997a). A second possibility (Fig. 10(II)) considers the anchorage of MyoA located at the IMC to the short actin filaments which would become joined to the plasma membrane through the aldolase complex according to Jewett and Sibley (2003). Because myosin–actin interactions take place on structures distributed in counter-clockwise arrangements such as the inner membrane complex, the subpellicular network and the subpellicular microtubules, the tachyzoites display gliding movements with twirls in a clockwise sense as also occurs during the penetration (Werk, 1985; Mondragon et al., 1994). Both these models should now be evaluated.

Acknowledgement We thank Biol. Olivia Reynoso for her technical support. We are grateful to Drs D. Kronberg and C. Mercier for their valuable comments on the manuscript. Micrographs were taken at the electron microscopy facility of CINVESTAVIPN, Mexico city. This work was supported by project CONACyT # 37713-N. Araceli Patro´n held the scholarship No 119829 from CONACyT.

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

References Allen, M.L., Dobrowolski, J.M., Muller, H., Sibley, L.D., Mansour, T.E., 1997. Cloning and characterization of actin depolymerizing factor from Toxoplasma gondii. Mol. Biochem. Parasitol. 88, 43–52. Aspenstrom, P., Karlsson, R., 1991. Interference with myosin subfragment1 binding by site-directed mutagenesis of actin. Eur. J. Biochem. 200, 35–41. Bannister, L.H., Mitchell, G.H., 1995. The role of the cytoskeleton in Plasmodium falciparum merozoite biology: an electron-microscopic view. Ann. Trop. Med. Parasitol. 89, 105–111. Bubb, M.R., Senderowickz, A.M.J., Sausville, E.A., Duncan, K.L.K., Korn, E.D., 1994. Jasplakinolide, a cytotoxic nature product, induces actin polymerization and completely inhibits the binding of phalloidin to F-actin. J. Biol. Chem. 269, 14869–14871. Carlier, M.F., Didry, D., Erk, I., Lepault, J., Pantaloni, D., 1994. Myosin subfragment-1-induced polymerization of G-actin. Formation of partially decorated filaments at high actin-S1 ratios. J. Biol. Chem. 269, 3829–3837. Chiappino, M.L., Nichols, B.A., O’Connor, R., 1984. Scanning electron microscopy of Toxoplasma gondii: parasite torsion and host cell responses during invasion. J. Protozool. 31, 288–292. Cintra, W.M., De Souza, W., 1985. Immunocytochemical localization of cytoskeletal proteins and electron microscopy of detergent extracted tachyzoites of Toxoplasma gondii. J. Submicrosc. Cytol. Pathol. 17, 503–508. Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W., Wingfield, P.T., 1995. In: Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W., Wingfield, P.T. (Eds.), Electrophoresis Current Protocols in Protein Science Current Protocols in Protein Science, vol. 1. Wiley, USA, pp. 10.5.1–10.5.11. De Souza, W., 1974. Fine structure of the conoid of T. gondii. Rev. Inst. Med. Trop. Sao Paulo 16, 32–38. Dobrowolski, J.M., Sibley, L.D., 1996. Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 84, 933–939. Dobrowolski, J.M., Carruthers, V.B., Sibley, L.D., 1997a. Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii. Mol. Microbiol. 26, 163–173. Dobrowolski, J.M., Niesman, I.R., Sibley, L.D., 1997b. Actin in the parasite Toxoplasma gondii is encoded by a single copy gene, ACT1 and exists primarily in a globular form. Cell Motil. Cytoskeleton 37, 253–262. Dubremetz, J.F., Torpier, G., 1978. Freeze fracture study of the pellicle of an Eimerian sporozoite (Protozoa, Coccidia). J. Ultrastruct. Res. 62, 94–109. Dunn, M.J., 1989. In: Harris, E.L.V., Angal, S. (Eds.), Determination of total protein concentration. Chapter 1. Initial Planning in Protein Purification Methods. A Practical Approach. Oxford University Press, pp. 10–18. Endo, T., Yagita, K., Yasuda, T., Nakamura, T., 1988. Detection and localization of actin in Toxoplasma gondii. Parasitol. Res. 75, 102–106. Etoh, S., Matsui, H., Tokuda, M., Itano, T., Nakamura, M., Hatase, O., 1990. Purification and immunohistochemical study of actin in mitochondrial matrix. Biochem. Int. 20, 599–606. Field, S.J., Pinder, J.C., Clough, B., Dluzewski, A.R., Wilson, R.J.M., Gratzer, W.B., 1993. Actin in the merozoite of the Malaria parasite, Plasmodium falciparum. Cell Motil. Cytoskeleton 25, 43–48. Giesemann, T., Schwarz, G., Nawrotzki, R., Berhorster, K., Rothkegel, M., Schluter, K., Schrader, N., Schindelin, H., Mendel, R.R., Kirsch, J., Jockusch, B.M., 2003. Complex formation between the postsynaptic scaffolding protein gephyrin, profilin, and Mena: a possible link to the microfilament system. J. Neurosci. 23, 8330–8339. Guerrero, A.L., Garcı´a, C.M., Villalba, J.D., Segura, M., Go´mez, C., Reyes, M.E., Herna´ndez, J.M., Garcı´a, R.M., De la Garza, M., 1996.

893

Actin-related proteins in Anabaena spp. and Escherichia coli. Microbiology 142, 1133–1140. Hakansson, S., Morisaki, H., Heuser, J., Sibley, L.D., 1999. Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion. Mol. Biol. Cell 10, 3539–3547. Hartwig, J.H., 1992. In: Carraway, K.L., Carraway, C.A.C. (Eds.), An ultrastructural approach to understanding the cytoskeleton Chapter 2. In the Cytoskeleton. A Practical Approach. Oxford University Press, USA, pp. 23–45. Herm-Gotz, A., Weiss, S., Stratmann, R., Fujita-Becker, S., Ruff, C., Meyhofer, E., Soldati, T., Manstein, D.J., Geeeves, M.A., Soldati, D., 2002. Toxoplasma gondii myosin A and its light chain: a fast, singleheaded, plus-end-directed motor. EMBO J. 21, 2149–2158. Hiroshi, M.J., Heuser, J.E., Sibley, L.D., 1995. Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J. Cell. Sci. 108, 2457–2464. Hu, K., Roos, D.S., Murray, J.M., 2002. A novel polymer of tubulin forms the conoid of Toxoplasma gondii. J. Cell. Biol. 156, 1039–1050. Huxley, H.E., 1963. Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. Mol. Biol. 7, 281–308. Jewett, J.T., Sibley, L.D., 2003. Aldolase forms a bridge between cell surface adhesins and the actin cytoskeleton in Apicomplexan parasites. Mol. Cell 11, 885–894. Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., Holmes, K.C., 1990. Atomic structure of the actin: DNase I complex. Nature 347, 37–44. King, C.A., 1988. Cell motility of sporozoan protozoa. Parasitol. Today 11, 315–318. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lazarides, E., Lindberg, U., 1974. Actin is naturally occurring inhibitor of deoxyribonuclease-I. Proc. Natl Acad. Sci. USA 71, 4742–4746. Lessard, J.L., 1988. Two monoclonal antibodies to actin: one muscle selective and one generally reactive. Cell Motil. Cytoskeleton 10, 349– 362. Mann, T., Beckers, C., 2001. Characterization of the subpellicular network, a filamentous membrane skeletal component in the parasite Toxoplasma gondii. Mol. Biochem. Parasitol. 115, 257–268. Mann, T., Gaskins, E., Beckers, C., 2002. Proteolytic processing of TgIMC1 during maturation of the membrane skeleton of Toxoplasma gondii. J. Biol. Chem. 277, 41240–41246. Meissner, M., Schluter, D., Soldati, D., 2002. Role of Toxoplasma gondii myosin A in parasite gliding and host cell invasion. Science 298, 837– 840. Mondragon, R., Frixione, E., 1996. Ca-Dependence of conoid extrusion in Toxoplasma gondii tachyzoites. J. Eukaryot. Microbiol. 43, 120–127. Mondragon, R., Meza, I., Frixione, E., 1994. Divalent cation and ATP dependent motility of Toxoplasma gondii tachyzoites after mild treatment with trypsin. J. Eukaryot. Microbiol. 41, 330–337. Moore, P.B., Huxley, H.E., De Rosier, D.J., 1970. Three-dimensional reconstruction of F-actin, thin filaments and decorated thin filaments. J. Mol. Biol. 50, 276–295. Morrissette, N.S., Murray, J.M., Roos, D.S., 1997. Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii. J. Cell. Sci. 110, 35–42. Nichols, B.A., Chiappino, M.L., 1987. Cytoskeleton of Toxoplasma gondii. J. Protozool. 34, 217–226. Oliveira-Lima, J.G., Mineo, J.R., Santos, A.A.D., Ferreira, G.L.S., Ferro, E.A.V., Oliveira, C.A., 2001. Improved methods for examination of Toxoplasma gondii cytoskeleton at ultrastructural level. Parasitol. Res. 87, 287–293. Opitz, C., Soldati, D., 2002. The glideosome: a dynamic complex powering gliding motion and host cell invasion by Toxoplasma gondii. Mol. Microbiol. 45, 597–604. Pardee, J.D., Spudich, J.A., 1982. Purification of muscle actin. Methods Enzymol. 85, 164–181.

894

A. Patro´n S et al. / International Journal for Parasitology 35 (2005) 883–894

Poupel, O., Tardieux, I., 1999. Toxoplasma gondii motility and host cell invasiveness are drastically impaired by jasplakinolide, a cyclic peptide stabilizing F-actin. Microbes Infect. 9, 653–662. Poupel, O., Boleti, H., Axisa, S., Couture-Tosi, E., Tardieux, I., 2000. Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments. Mol. Biol. Cell 11, 355–368. Russell, D.G., Burns, R.G., 1984. The polar ring of coccidian sporozoites: a unique microtubule-organizing centre. J. Cell. Sci. 65, 193–207. Russell, D.G., Sinden, R.E., 1981. The role of the cytoskeleton in the motility of coccidian sporozoites. J. Cell. Sci. 50, 345–359. Ryning, F.W., Remington, J.S., 1978. Effect of cytochalasin D on Toxoplasma gondii cell entry. Infect. Immun. 20, 739–743. Schatten, H., Sibley, L.D., Ris, H., 2003. Structural evidence for actin-like filaments in Toxoplasma gondii using high-resolution low-voltage field emission scanning electron microscopy. Microsc. Microanal. 9, 330–335. Schwartzman, J.D., Pfefferkorn, E.R., 1983. Immunofluorescent localization of myosin at the anterior pole of the coccidian, Toxoplasma gondii. J. Protozool. 30, 657–661.

Shaw, M.K., Tilney, L.G., 1999. Induction of an acrosomal process in Toxoplasma gondii: visualization of actin filaments in a protozoan parasite. Proc. Natl Acad. Sci. USA 96, 9095–9099. Sheterline, P., Clayton, J., Sparrow, J.C., 1998. Actin, fourth ed. Oxford University Press, New York. Thulasiraman, V., Ferreyra, R.G., Frydman, J., 2000. Monitoring actin by affinity chromatography on immobilized DNAseI using formamide as eluant. Eur. J. Biochem. 110, 343–348. Tsuyama, S., Takenaka, S., 1997. Relationships between cytoskeletal proteins and the toxic effects of drugs. J. Toxicol. Sci. 22, 383–395. Werk, R., 1985. How does Toxoplasma gondii enter host cells? Rev. Infect. Dis. 7, 449–457. Yasuda, T., Yagita, K., Nakamura, T., Endo, T., 1988. Immunocytochemical localization of actin in Toxoplasma gondii. Parasitol. Res. 75, 107–113. Zechel, K., 1980. Isolation of polymerization-competent cytoplasmic actin by affinity chromatography on immobilized DNAse I using formamide as eluant. Eur. J. Biochem. 110, 343–348.