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2 Laboratoire de Biologie des Protistes, Centre National de la Recherche Scientifique (CNRS), UMR 6023, Université Blaise Pascal, 63177 Aubière, France
3 Department of Molecular Cell Research, Max Planck Institute for Medical Research, D-69120 Heidelberg, Germany
4 Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom
5 UMR CNRS 8576 Université des Sciences et Technologies de Lille, France
Address correspondence to Dr. Dominique Soldati, Department of Biological Sciences, Imperial College of Science, Technology, and Medicine, Imperial College Road, London SW7 2AZ, United Kingdom. Tel.: (44) 207-594-5342. Fax: (44) 207-584-2056. E-mail: d.soldati{at}ic.ac.uk
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Abstract |
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Key Words: Apicomplexa; unconventional myosin XIV; localization; Toxoplasma gondii; cell division
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Introduction |
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Here, we report about the determination of the expression pattern of MyoB/C products and the predominant role of their tails in subcellular localization. The cellular distribution and phenotypes resulting from the overexpression of wild-type or mutant forms of MyoB and MyoC strongly suggest that the MyoB/C gene products are implicated in parasite division.
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Results |
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Toward the identification of a myosin contributing to cell division, we analyzed the pattern of expression of the five known myosins in the two invasive life stage forms of T. gondii present in intermediate hosts. Specific transcripts coding for myosins A, B, C, and D (not shown), but not E, were amplified by RT-PCR using total RNAs isolated from the rapidly replicating tachyzoites (Fig. 2 A). Furthermore, a comparison of the amount of specific transcripts between tachyzoites and the dormant, encysted bradyzoites was assessed by semiquantitative RT-PCR (Fig. 2 B), as previously described (Yahiaoui et al., 1999). Rather unexpectedly and with the exception of MyoA, the myosin mRNAs were more abundant in the persistent stage. This analysis did not distinguish between the increased rate of transcription and the increased stability of transcripts in bradyzoites compared with tachyzoites. Expressed sequence tag (EST) clones corresponding to myosins are extremely underrepresented in the 10,000 ESTs found in the T. gondii database (Ajioka et al., 1998). Nevertheless, the same tendency toward a predominant representation in bradyzoite compared with tachyzoite cDNAs was observed (http://www.cbil.upenn.edu/ParaDBs/). Indeed, 11 EST clones specific for MyoC were present in the in vivo bradyzoite cDNA library compared with two ESTs in the much larger pool of T. gondii RH strain tachyzoite ESTs. This imbalance reflects and strengthens the results obtained by RT-PCR. The two transcripts encoding MyoB and MyoC were previously described and presumed to derive from alternative RNA splicing (Heintzelman and Schwartzman, 1997).
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Transient expression of MyoB/C gene products reveals their biased segregation between daughter cells after endodyogeny
The very low expression level of MyoB in tachyzoites and the difficulty to unambiguously determine the subcellular localization of the two alternatively spliced products prompted us to adopt an epitope tagging strategy, as used for the characterization of MyoA (Hettmann et al., 2000). In addition, transient expression of MyoB, MyoC, and the MyoB/C motor domain lacking the tail (MyoB/Ctail) revealed unexpected features.
First, a striking asymmetry in the repartition of MyoB, MyoC, and even MyoB/Ctail in daughter cells was apparent 24 and 48 h after transfection. Freshly lysed parasites are asynchronous and thus undergo replication at different times after transfection and host cell infection, resulting in variable numbers of strongly positive and almost negative parasites per vacuole. Likely, if the transfected parasites divided before the transgene products had accumulated, the myosins would be present in both daughter cells. In contrast, if the protein accumulated before division, it could not be distributed evenly between the daughter cells (Fig. 4 A D in red, E and F in green). The anti-TgMIC6 was used as a control to detect all the parasites present in the vacuole. Other cotransfected cytoplasmic or organellar markers (green fluorescence protein [GFP], catalase, or MIC6) were, without exception, evenly distributed among the parasites of a given vacuole (unpublished data). The only other reported segregation defect in T. gondii was observed after the expression of a chimeric molecule targeted to the apicoplast, which prevented replication of the organelle in daughter cells (He et al., 2001).
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Although parasites stably expressing MyoB and MyoC were obtained and analyzed (see below), multiple attempts to generate stable transformants expressing MyoB/Ctail remained unsuccessful, suggesting that the truncated form of the protein was not tolerated.
MyoB and MyoC exhibit distinct cellular localizations
To analyze more closely the localization of the MyoB/C gene products in nondividing parasites, we generated recombinants stably expressing mycMyoB or mycMyoC. We confirmed the expression of polypeptides of the anticipated sizes by Western blotting with anti-myc antibodies (Fig. 5 C). The stably transformed parasites were then examined by immunofluorescence assay (IFA), revealing distinct, subcellular steady-state distributions (Fig. 5, A and B). As previously observed in transient transfections, MyoB was spread throughout the cytoplasm associated with a punctate structure, whereas the distribution of MyoC was mostly restricted to the posterior and weakly to the anterior poles of the parasites (Fig. 5 E). Double immunofluorescence staining of MyoB and MyoA showed no major overlap. Additionally, the double staining against MyoC and the apical micronemal protein TgMIC6 confirmed the predominant posterior localization of MyoC. High resolution confocal microscopy distinctly identified the staining of MyoC as a ring structure, likely corresponding to the posterior polar ring and more weakly to the apical polar ring at the terminal regions of the IMC. A schematic representation of a tachyzoite cytoskeleton based on previous EM studies (Nichols and Chiappino, 1987) is depicted in Fig. 5 D. At the apical pole, the conoid is associated with the apical polar ring, the IMC, and the sets of 22 pellicular microtubules. The posterior polar ring delineates the termination of the IMC at the rear end of the parasite. A large parasitophorous vacuole containing 32 parasites arranged in rosette illustrates the focused staining at the posterior pole of the parasites, which corresponds to the center of the rosette. A weaker staining at the periphery corresponds to the apical tip of each parasite (Fig. 5 E). The restricted localization of MyoC is unlikely to depend only on direct interactions with actin filaments since the treatment of recombinant parasites with 10 µM cytochalasin D for 2 h did not alter the ring structures visualized by IFA (unpublished data).
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Discussion |
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Parasites treated with BDM, an inhibitor of myosin ATPase, underwent proper nuclear replication but showed impairment in cell division, similar to the cytokinesis defects observed in other organisms (unpublished data). This observation suggested a critical role for a myosin during endodyogeny, but was inconclusive because this drug is known to have pleiotropic targets. Until now, no myosin of class II or V has been identified in Apicomplexa. To assign which myosin could be involved in the cell division process, we examined the pattern of expression and subcellular distribution of the five class XIV myosins identified so far in T. gondii. MyoA is the prominent motor candidate for powering parasite gliding motility, whereas MyoE could not be involved because it is not expressed at a detectable level in tachyzoites. We confidently excluded MyoD because disruption of the MyoD gene by homologous recombination did not affect parasite division (unpublished results).
MyoB and MyoC are the products of alternatively spliced transcripts from the same gene (Fig. 3; Heintzelman and Schwartzman, 1997). These two transcripts are present in tachyzoites but are upregulated in bradyzoites. The abundance of the respective myosins suggests that a partial splicing leading to the production of MyoB occurs much less frequently. Thus, MyoC appears to be the predominant product in tachyzoites. This observation was confirmed by the fusion of a GFP coding sequence to a fragment of the MyoB/C gene covering the tail exons, which generates essentially GFPMyoCtail.
Unexpectedly, MyoB, MyoC, and MyoB/Ctail (including the motor and a divergent IQ motif) have a dominant effect under conditions of transient expression; this motor does not segregate equally during cell division. There is no obvious explanation for this unusual behavior, but it clearly implies an interaction of the motor with an as yet undefined cellular structure that might fail to segregate properly due to the overexpression or is naturally unevenly distributed between the daughter cells. We speculate that this structure or organelle might be the IMC or part thereof. First, during transient expression, MyoB, MyoC, and even the motor domain truncated of its tail localize to the cell periphery, to punctate structures and patches, as well as to membrane structures that appear to extend between the dividing daughter cells. Second, the almost exclusive localization of MyoC at the termination of the IMC suggests that this myosin might fulfill a direct role in assembly and elongation of this complex during budding of the daughter cells. Indeed, MyoC is associated with the IMC at an early stage of daughter cell formation (Fig. 6 D). This is reminiscent of the situation in Caenorhabditis elegans where myosin VI was shown to be required for asymmetric segregation of cellular components during spermatogenesis (Kelleher et al., 2000). The partitioning of MyoC with the detergent-insoluble fraction implies an interaction with components of the parasite cytoskeleton. This posterior structure is not disrupted by treatment with cytochalasin D and thus appears independent of the presence of intact actin filaments. Nevertheless, MyoC was previously shown to be a classical myosin, binding to F-actin in an ATP-sensitive fashion (Heintzelman and Schwartzman, 1999). The analysis of GFPCtail fusion confirmed that the localization and sedimentation determinants are confined within its tail domain.
In addition to the segregation defects, transient expression of both MyoB and MyoC leads to the accumulation of residual bodies at the posterior of the parasites after division. Both morphological defects observed during transient expression of MyoC and MyoB/Ctail could not be analyzed further in stable cell lines because no viable transformant could be obtained with MyoB/C
tail, and lines only expressing low levels of MyoC were obtained. Overexpression of class I myosins in Dictyostelium discoideum has been reported to lead to a phenotype similar to knockout mutants (Novak and Titus, 1997). As the MyoB/C gene is most likely indispensable, we will need to generate a conditional knockout based on an inducible system, which has recently been established for T. gondii (Meissner et al., 2001).
Here, stable MyoB overexpression induced a clear defect in proper separation of the daughter cell membranes, although nuclear division and formation of the daughter conoids appeared normal. In addition, the mutant did not show any impairment in host cell invasion (unpublished data), another actomyosin-dependent process proposed to involve MyoA. Generation of large residual bodies at the posterior end of the parasites and the presence of bleb-like structures randomly distributed around the parasites suggest an impairment in endodyogeny. Residual bodies appear to concentrate MyoB and actin but are essentially devoid of organelles and DNA. These bodies are therefore distinct from the residual bodies induced by treatment with actin inhibitors, which were shown to contain rhoptries, micronemes, a part of the mitochondrion, the apicoplast, and some ER (Shaw et al., 2000). The actomyosin system thus seems to play a role late in the division process, after replication, when the IMC (partly of the mother cell, partly formed de novo) slides over the newly formed conoid and microtubule basket of the daughter cells.
MyoC is the best candidate for directly participating late in the division process, as it accompanies the posterior polar ring during its descent through the mother cell, encircling each daughter. This role is reminiscent of the proposed function of myosin VI in the sliding of membranes along the length of the spermatid nuclei and axonemal microtubules during the individualization of syncitial spermatids in Drosophila (Hicks et al., 1999). The phenotypic consequences of MyoB overexpression are also suggestive of its role at the end of cell division. In this case, the sliding of the mother plasma membrane onto the IMC of the budding daughter cells does not occur properly and tightly, leading to the creation of plasma membrane blebs. MyoB is associated with the cell periphery and IMC lamellae, suggesting its direct involvement. Additionally, the creation of large residual bodies at the posterior pole are indicative of an improper sealing or closure, a process analogous to the constriction of the cleavage furrow occurring in other eukaryotic cells, which is myosin II dependent. We conclude that both MyoB and MyoC have localizations relevant to parasite division, but the similar phenotype caused by overexpression of MyoB, MyoC, and MyoB/Ctail is more likely to result from an interference with the function of the predominant product in tachyzoites, MyoC.
Although close homologues of T. gondii MyoA can be found in all apicomplexan parasites for which significant DNA sequence information is available, including Plasmodium, Neospora, Eimeria, and Cryptosporidium species, there is no evidence for the presence of a myosin closely related to MyoB/C in other apicomplexan parasites thus far. Endodyogeny shares many similarities with the general process of shizogony, the usual asexual multiplication mechanism in Apicomplexa; however, endodyogeny takes place in a fully differentiated mother parasite that keeps its highly sophisticated invasion complex throughout division (Porchet-Hennere et al., 1985). In particular, the IMC, which is composed of Golgi-derived saccules, is maintained in the mother and partly recycled into the daughter cells by a process that is poorly understood. Contrasting to this, in other Apicomplexa such as Plasmodium, zoites form de novo at the periphery of a multinucleate, undifferentiated cytoplasmic mass. Therefore, T. gondii may have evolved specific ways of integrating old material into new zoites, and because this process is highly dynamic in terms of membranecytoskeleton interactions, myosins are likely to be involved.
Finally, whereas intraperitoneal infection with RH is always lethal, impairment in cell division caused by overexpression of MyoB leads to a major reduction in virulence and the formation of cysts in the brain of infected mice. It will be very interesting to examine the ultrastructure of these cysts, but their limited numbers has hampered such analysis so far. These results are very similar to the ones reported for the knockout of the dense granule protein 2 gene (GRA2) in RH, which led to decreased virulence and chronic infection (Mercier et al., 1998).
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Materials and methods |
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Growth of parasites and isolation of DNA and RNA
T. gondii tachyzoites (RH strain wild-type and RH hypoxanthine-xanthine-guanine-phosphoribosyltransferase [hxgprt]-) were grown in human foreskin fibroblasts (HFF) maintained in DME with 10% FCS, 2 mM glutamine, and 25 µg/ml gentamicin. Parasites were harvested after host cell lysis and purified by passage through filters with 3.0 µm pores and centrifugation in PBS. Genomic DNA was isolated from purified parasites as previously described (Sibley and Boothroyd, 1992). Total RNAs were prepared using "RNA clean2" (AGS GmbH) according to the manufacturer's instructions.
T. gondii genomic library screening
A clone containing the MyoB/C locus was isolated from a cosmid library made in a SuperCos vector modified with a SAG1/ble T. gondii selection cassette. The library (provided by D. Sibley and D. Howe, Washington University, St. Louis, MO) was prepared from a Sau3AI partial digestion of RH genomic DNA ligated into the BamHI cloning site. We partially sequenced the MyoB/C locus (GenBank/EMBL/DDBJ accession number AAF09586). Probes were labeled using DIG-11-dUTP. Hybridization and chemiluminescent CSPD detection were performed according to the manufacturer (Roche Molecular Biochemicals).
Construction of T. gondii expression vectors
The MyoB and MyoC coding sequences were amplified by RT-PCR using total RNA from the Prugniaud strain. Both cDNAs were sequenced (GenBank/EMBL/DDBJ accession numbers MyoB, AF438184; MyoC, AF438183). The vectors pTmycMyoB and pTmycMyoC were generated by cloning the cDNA between NsiI and PacI sites of the pTmycGFP-HX vector (Hettmann et al., 2000). The restriction sites PstI and PacI were introduced in the sense primer 1B/C and antisense primer 2B or primer 2C, respectively. Both myosins carry an NH2-terminal c-myc epitope and 7xHis residues (MQEQKLISEEDLAMAMHHHHHHH) to produce mycMyoB and mycMyoC. The vector pTmycMyoB/Ctail was generated by the PCR amplification of a fragment coding for the first 754 amino acids of MyoB/C using primers 1B/C and 2B/C. The mycGFPtail fusion vectors were constructed by cloning the tail fragments between PstI and PacI sites of the pTGFP-HX vector. To generate pTGFPtailB and pTGFPtailC, the PstI-PacI fragments corresponding to the COOH-terminal tails of MyoB and MyoC were amplified from cDNAs using primers 3B/C and 2B or 3B/C and 2C. A GFP fusion with the last 118 amino acids of MyoC was obtained by PCR amplification using primers 4C and 2C to generate pTGFPtailC
118. A fusion with the genomic sequence of MyoB/C tail was obtained by PCR amplification from a cosmid clone using primers 3B/C and 2C to generate pTGFPtailB/C. The 3'UTR of MyoB/C was amplified from a cosmid clone using primers 5C and 6C and introduced into pTGFPtailB/C to replace the 3' untranslated region (UTR) of SAG1.
Primers:1B/C, 5'-AACTGCAGGACACGCAGCTGGAACTCGAG-3'; 2B, 5'-GCCGGATCCTTAATTAACTATGTTTTTGATTATGTTTCCATGTCAG-3'; 2C, 5'-GCCGGATCCTTAATTAAGGTCTATCCGGCGCACAGGC-3'; 2B/C, 5'-GCCTTAATTAAGATGCATGTCTGCGCGCGTTTGATTCC-3'; 3B/C, 5'-GCCGGATCCGCTGCAGTCCTGGGCCCGATGTGG-3'; 4C, 5'-AACTGCAGGCATTGTGCGCGTCATGAATAGC-3'; 5C, 5'-CCTTAATTAACCTTTAAAGTGGACAAGGGTGA-3'; 6C, 5'-GCCGGATCCAGGACGGTAGTGGTCGGG-3'.
Parasite transfection and selection of stable transformants
T. gondii tachyzoites (RHhxgprt-) were transfected by electroporation as previously described (Soldati and Boothroyd, 1993). The HXGPRT was used as a positive selectable marker in the presence of mycophenolic acid and xanthine as previously described (Donald et al., 1996).
Western analysis of parasite lysates
Crude extracts of tachyzoites were separated by SDS-PAGE (Laemmli, 1970). Western blot analysis was performed essentially as described by Soldati et al. (1998) using 810% polyacrylamide gels run under reducing conditions, followed by transfer to Hybond ECL nitrocellulose. For detection, affinity-purified, HRP-conjugated goat antimouse IgG or goat antirabbit IgG (1:2,000) and the ECL system (Amersham Pharmacia Biotech) were used. Direct recording of chemoluminescent signals and densitometry by the Luminescent Image Analyzer LAS-1000 (FujiFilm) allowed for quantification of signal intensities within a broad linear range.
Cell fractionation
109 parasites, freshly released from infected cells, were resuspended in 1 ml of bufffer (either PBS, PBS/1 M NaCl, PBS/0.1 M Na2CO3 (pH 11.5), or PBS/2% Triton X-100) and lysed by sonication (four times, 30 s each; on ice). 10 mM ATP was freshly added to minimize the interaction of the myosin with F-actin. Pellet and soluble fractions were separated by ultracentrifuging for 1 h at 55,000 rpm at 4°C.
Indirect immunofluorescence microscopy and detection of GFP in T. gondii
All manipulations were performed at room temperature. Intracellular parasites grown in HFF on glass slides were fixed with 4% paraformaldehyde and 0.05% glutaraldehyde for 20 min. After fixation, slides were rinsed in PBS/0.1 M glycine. Cells were then permeabilized in PBS/0.2% Triton X-100 for 20 min and blocked in the same buffer with 2% FCS. Slides were incubated for 60 min with primary antibodies diluted in PBS/1% FCS, washed, and incubated for 60 min with Alexa488- or FITC-labeled goat antimouse IgGs diluted in PBS/1% FCS. Rapid freezing in ultra cold methanol with combined fixation/permeabilization was performed as described by Neuhaus et al. (1998). Slides were mounted in Vectashield and kept at 4°C in the dark. Intracellular parasites expressing GFP were fixed according to the above protocol and mounted immediately. Confocal images were collected with a Leica laser scanning confocal microscope (TCS-NT DM/IRB) using a 100x Plan-Apo objective with NA 1.30. Single optical sections were recorded with an optimal pinhole of 1.0 and 16 times averaging. All other micrographs were obtained on a Zeiss Axiophot with a camera (Photometrics Type CH-250). Adobe Photoshop and Canvas 7 were used for image processing.
EM
HFF cells were plated on sapphire coverslips for 3 d before infection with parasites. Then, the coverslips were plunged into an ethane slush at -170°C, freeze substituted, and embedded in Lowicryl as previously described (Neuhaus et al., 1998). Sections were observed and documented on a Philips 400 T TEM. The EM negatives were scanned at 1,200 dpi and imported into Adobe Photoshop for processing.
In vivo experiments
BALB-c mice were inoculated by intraperitoneal injection of 20 freshly harvested tachyzoite parasites from the wild-type RH strain or with MyoB-overexpressing RH. 3 wk after infection, the number of survivors was recorded and their T. gondii serology was tested by Western blotting against an RH tachyzoite lysate.
Isolation of T. gondii cysts from the brain of infected mice
Seropositive mice survivors were killed 8 wk after intraperitoneal injection of 20 parasites from MyoB-expressing RH. Parasite cysts were isolated from the brain as previously described (Tomavo et al., 1991). The cyst preparation was incubated for 1 h with the FITC-labeled lectin from Dolichos biflorus, diluted 1:50, at 37°C.
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Footnotes |
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D. Soldati, T. Soldati, and R. Stratmann's present address is Department of Biological Sciences, Imperial College of Science, Technology, and Medicine, London SW7 2AZ, United Kingdom.
* Abbreviations used in this paper: BDM, 2,3-butanedione monoxime; EST, expressed sequence tag; GFP, green fluorescence protein; HFF, human foreskin fibroblast; HXGPRT, hypoxanthine-xanthine-guanine-phosphoribosyltransferase; IFA, immunofluorescence assay; IMC, inner membrane complex; UTR, untranslated region.
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Acknowledgments |
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This work was funded by the Deutsche Forschungsgemeinschaft (DFG grant SO 366/1-1, SO366/1-2) and The Biotechnology and Biological Sciences Research Council. Dr. F. Delbac was supported by a grant of the Von Humboldt foundation. E. Neuhaus was a recipient of a postdoctoral fellowship from the Max Planck Society.
Submitted: 28 December 2000
Revised: 28 September 2001
Accepted: 5 October 2001
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