ARTICLE |
Correspondence to: Annie Bonhomme, UPRES EA 2070, IFR, 53-51 rue Cognacq Jay, 51095 Reims Cedex, France. E-mail: annie.bonhomme@univ-reims.fr
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Summary |
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The gliding motility of the protozoan parasite Toxoplasma gondii and its invasion of cells are powered by an actinmyosin motor. We have studied the spatial distribution and relationship between these two cytoskeleton proteins and calmodulin (CaM), the Ca2+-dependent protein involved in invasion by T. gondii. A 3D reconstruction using labeling and tomographic studies showed that actin was present as a V-like structure in the conoidal part of the parasite. The myosin distribution overlapped that of actin, and CaM was concentrated at the center of the apical pole. We demonstrated that the actomyosin network, CaM, and myosin light-chain kinases are confined to the apical pole of the T. gondii tachyzoite. MLCK could act as an intermediate molecule between CaM and the cytoskeleton proteins. We have developed a model of the organization of the actomyosinCaM complex and the steps of a signaling pathway for parasite motility.
(J Histochem Cytochem 49:445453, 2001)
Key Words: T. gondii, calmodulin, actomyosin complex, MLCK, confocal microscopy, 3D reconstruction
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Introduction |
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THE COCCIDIAN Toxoplasma gondii, belonging to the phylum Apicomplexa, can infect almost any warm-blooded vertebrate. It is a major cause of severe congenital disease in humans and is one of the main opportunistic pathogens in AIDS. T. gondii is an obligate intracellular parasite and so must be able to enter the host cell. The invasion process of T. gondii differs from endocytotic uptake, and penetration is an active function of the tachyzoite (the invasive stage of T. gondii). The parasite has no specialized structures for movement, and motility and invasion are powered by the cytoskeleton. T. gondii is highly polarized and has a complex cytoskeleton with an apical cone-like structure or conoid connecting to 22 singlet microtubules that extend two thirds of the length of the parasite (
As we have recently demonstrated (
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Materials and Methods |
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Parasites
Tachyzoites of the T. gondii RH strain were maintained by serial passages at 34-day intervals in a culture of THP1 cells (myelomonocytic cell line; American Type Culture Collection, Rockville, MD, non-adherent cells) in RPMI medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Tachyzoites were removed when the THP1 cells were lysed, centrifuged at 500 x g for 15 min, and suspended in RPMI medium.
Cells
KB cells (human epidermoid carcinoma epithelium cell line 86103004) were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, and streptomycinpenicillin (100 µg/ml100 U/ml) at 37C in an atmosphere of saturated 5% CO2/95% air. The cells were harvested by trypsination (0.05% trypsin0.02% EDTA) and seeded at 5.105 cells/25-ml flask.
Infection of KB Cells
KB cells were plated on coverslips (104 cells) for 48 hr to obtain a subconfluent culture. The coverslips were then placed in 24-well microtiter plates (Nunc; Polylabo Block, Strasbourg, France) containing 1 ml RPMI per well. The monolayers of KB cells were then infected with washed tachyzoites with a cell:parasite ratio of 1:10 and were left in contact for 2496 hr.
Fluorescent Immunolabeling of Actin
Extracellular tachyzoites were placed on glass slides. Intracellular tachyzoites in their KB host cells plated on coverslips were treated by permeabilizing the host cell membrane by immersion for 3 min in 0.1% Triton X-100 and then fixed in 3% paraformaldehyde in PBS, pH 7.2, for 30 min. The cells were first incubated for 30 min with PBS containing 0.2% gelatin, 3% BSA, and then with a rabbit polyclonal anti-actin (chicken back muscle) antibody (Chemicon; Euromedex, Souffelweyersheim, France) diluted 1:30 in the same buffer for 1 hr at room temperature (RT). Tested in Western blotting with tachyzoite homogenate, this antibody recognized particularly a band around 40 kD. The samples were then washed six times with PBSgelatinBSA and incubated with biotinylated donkey anti-rabbit IgG antibody (diluted 1:50 in the same buffer) for 1 hr at RT. Cells were washed three times in PBSgelatinBSA and three times in PBS and then incubated with streptavidinTexas Red (diluted 1:50 in PBS) for 15 min in the dark, and washed again with PBS. Their nuclei were stained with 150 mM MgCl2 and 100 µM chromomycin A3 for 15 min in the dark, and finally with MgCl2. The cells were treated with Citifluor and examined by confocal microscopy (microscope Zeiss Axioplan, confocal part: MRC 600).
Fluorescent Immunolabeling of Myosin
Myosin was labeled in the same way as actin, except that the cells were first fixed using 3% paraformaldehyde before permeabilization with 0.1% Triton X-100. The primary antibody was a rabbit polyclonal anti-(bovine uterus smooth muscle) myosin antibody (diluted 1:50) (Valbiotech; Paris, France). This antibody recognized particularly two bands at 120 kD and 110 kD and a more intense band at 90 kD when tested by Western blotting with a tachyzoite homogenate. These seem to correspond to TgM-C, TgM-B, and TgM-A.
Fluorescent Immunolabeling of Calmodulin
This experiment was performed using the myosin protocol, except that the primary antibody was a mouse monoclonal anti-(Dictyostelium, bovine, rat, and chicken) calmodulin antibody (Tebu; Le Perray en Yvelines, France) diluted 1:80 (Amersham; Les Ulis, France) and the secondary antibody was a biotinylated sheep anti-mouse IgG antibody diluted 1:50 (Amersham).
Fluorescent Immunolabeling of Myosin Light-chain Kinases
MLCK was labeled according to the CaM labeling protocol. The primary antibody was a mouse monoclonal anti-chicken MLCK antibody (Sigma; St Quentin Fallavier, France) diluted 1:50 and the secondary antibody was a biotinylated sheep anti-mouse IgG antibody diluted 1:50 (Amersham.
Controls without the primary antibody were prepared in four experiments.
Double CalmodulinActin Immunolabeling in Extracellular Tachyzoites. The main problem with this experiment was to balance the detergent extraction before fixation (for the cytoskeleton protein) against calmodulin removal (calmodulin being a soluble protein). We used extraction for 1 min before fixation to keep the CaM in place. We first labeled the CaM using the protocol used for CaM immunodetection alone (see above). Actin was then labeled by incubating the samples with rabbit polyclonal anti-actin (diluted 1:30), then with a digoxigenin sheep anti-rabbit IgG F(ab')2 fragment (diluted 1:100) for 1 hr, and finally with a fluorescein sheep anti-digoxigenin Fab fragment (diluted:150) for 30 min.
Double CalmodulinMyosin Immunolabeling in Extracellular Tachyzoites. Cells were fixed with 3% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and CaM was immunolabeled, followed by myosin. Labeling was performed according to the CaMactin double-labeling protocol.
Confocal Microscopy, Optical Sections, and 3D Reconstruction
Specimens were examined with an MRC 600 (BioRad; Richmond, CA) confocal laser scanning microscope equipped with two lasers (argon and heliumneon) mounted on a Zeiss Axioplan microscope (Zeiss; Thornwood, NY). The Texas red fluorophore was visualized with the 543-nm excitation wavelength of a heliumneon laser and the chromomycin A3 with the 457-nm excitation wavelength of an argon laser. Images were preprocessed with the Comos software package (BioRad) to increase the contrast and to merge the two labelings. Photomicrographs of the double labeling (proteinnucleus) were obtained by direct visualization, which corresponds to the fluorescence emission from one plane of the object. Serial optical sections (around 20) (Z-series) were processed on specimens at 0.2-µm steps. Three-dimensional reconstruction was performed by converting the 2D images of a Z-series into a volume in which we could make virtual cuts of the reconstructed labeling. The 3D reconstruction was performed with Analyze software (Analyze Mayo Bir; CN Software, Southwater, West Sussex, UK) on a Sun Microsystems workstation (Sun Microsystems; Velizy, France). This software makes it possible to move the 3D-reconstructed objects around and to present the most informative viewing angles.
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Results |
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Localization of Actin, Myosin, Calmodulin, and Myosin Light-chain Kinases of T. gondii
Nuclei stained with chromomycin A3 appeared as green fluorescence. The apical pole of the parasite was at the opposite end from the nucleus.
Actin. Actin was mainly located in the anterior third of the parasite, with a circumferential pattern beneath the parasite membrane complex. The labeling at the apical end appeared to lie under the membrane with a specific V structure (Fig 1A1). The intracellular parasites had similar staining at their apical end (Fig 1A2).
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Myosin. Extracellular T. gondii were diffusely stained with the rabbit polyclonal anti-myosin antibody in the broad anterior third of the parasite, with the greatest intensity at the periphery of the conoid (Fig 1B1). Intracellular parasites (Fig 1B2) had a similar myosin distribution.
Calmodulin. Most of the CaM immunostaining was at the pole opposite the green nucleus, the apical end (Fig 1C1). The CaM in intracellular tachyzoites (Fig 1C2) was always prominent at the apical end, but was also distributed beneath the length of the membrane complex of the parasites (arrowed).
Myosin Light-chain Kinases. The red fluorescence was less intense but essentially at the apical pole of the extracellular parasite; it was redistributed in the cytosol of the intracellular parasites (Fig 1D1 and 1D2).
Examination of the controls (without primary antibody) showed no labeling, except for the nuclei stained with chromomycin A3.
Extracellular parasites stained with antibodies to actin (Fig 2A1) and CaM (Fig 2A2) showed both CaM and actin at the anterior pole of the tachyzoites. The specific V structure seen in the single labeling was not observed, probably because of the treatment used for the double immunolabeling (the rapid extraction before fixation needed to keep CaM in place made it impossible to detect the V structure).
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Fig 2B shows the labeling for myosin (Fig 2B1) and CaM (Fig 2B2), with both proteins at the apical end of the extracellular parasites.
Superimposition of the two images (Fig 2A3 and 2B3) showed a co-localization (actincalmodulin, myosincalmodulin) which appeared in yellow fluorescence at the apical extremity of the parasites.
Reconstruction of Optical Sections and Visualization of the Labeling
The optical sections of a Z-series were reconstructed to form the volume of the labeled object. The numbers in Fig 3 give an overall view of the reconstructed labeling (1), and a slice (2) in the volume to show the internal part of the object labeling. Orthosections were prepared (4) from a single optical section (3) according to X and Y axes.
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The 3D reconstruction of the actin labeling in the extracellular parasites (Fig 3A1) confirmed the apical concentration of this protein, as well as the circumferential actin distribution over the entire tachyzoite with "bow structures" (arrows). The apical end of the parasite appeared to be "hooded" by this cytoskeleton protein. A slice through the object (Fig 3A2) revealed that most of the apical actin labeling was in the periphery, with the specific V structure (arrows), without inner labeling. We conclude that T. gondii actin lies below the membrane. The single optical section (Fig 3A3) and orthosections in the anterior pole along the X and Y axes (Fig 3A4) gave the same circumferential distribution of actin (as a ring) with punctate staining.
The overall 3D visualization of myosin (Fig 3B1) suggested that it was concentrated at the anterior pole of the parasite with a more diffuse pattern. A slice (Fig 3B2) showed differences in the gray scale labeling of intensity in the apical staining, indicating that myosin is distributed along a concentration gradient, with pronounced labeling at the periphery (light gray scale) and weaker labeling in the center (dark gray scale). Orthosections confirmed the diffuse labeling of myosin with a circumferential accumulation (Fig 3B3 and 3B4)
Calmodulin (Fig 3C1) was concentrated in the apical end of the parasite. A slice through the global volume (Fig 3C2) showed that the apical CaM labeling was compact and entire. The resulting CaM concentration gradient showed most CaM in the center, inside the apical end (light gray scale) and less towards the outside. This was supported by the XY orthosections showing CaM staining in the shape of an intense full sphere (Fig 3C3 and 3C4).
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Discussion |
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The actomyosin system generates biochemical forces for cell movement and cytokinesis. The motility of T. gondii is critically dependent on actin filaments and is driven by a myosin motor. Previous studies using heterologous antisera or actin-specific antibodies have indicated that actin is essentially confined to the apical end of the T. gondii tachyzoite (
The CaM inhibitors calmidazolium and trifluoperazine significantly reduce the parasite invasion index in vitro (
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Acknowledgments |
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We thank Hervé Kaplan and Francis Deligny for technical assistance and Dr Owen Parkes for revising the English text.
Received for publication October 17, 2000; accepted October 18, 2000.
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