Functional Dissection of the Apicomplexan Glideosome Molecular Architecture

SummaryThe glideosome of apicomplexan parasites is an actin- and myosin-based machine located at the pellicle, between the plasma membrane (PM) and inner membrane complex (IMC), that powers parasite motility, migration, and host cell invasion and egress. It is composed of myosin A, its light chain MLC1, and two gliding-associated proteins, GAP50 and GAP45. We identify GAP40, a polytopic protein of the IMC, as an additional glideosome component and show that GAP45 is anchored to the PM and IMC via its N- and C-terminal extremities, respectively. While the C-terminal region of GAP45 recruits MLC1-MyoA to the IMC, the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress. The orthologous Plasmodium falciparum GAP45 can fulfill this dual function, as shown by transgenera complementation, whereas the coccidian GAP45 homolog (designated here as) GAP70 specifically recruits the glideosome to the apical cap of the parasite

Dominique Soldati-Favre: Bringing Toxoplasma gondii to the Molecular World

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A. Schematic representation of a <i>T</i>. <i>gondii</i> tachyzoite highlighting the localization and composition of the three glideosomes.

<p>B. Localization of three glideosomes by immunofluorescence using the endogenously tagged TgGAP70 and TgGAP80 and the anti-TgGAP45 antibodies. Scale bars: 2 μm. C. Relocalization of MycMyoC to the periphery of the tachyzoite in addition to its basal localization in absence of TgMyoA in the MycMyoC-cKD/MyoA-KO strain. Scale bars: 2 μm.</p

Summary of the phenotype and adaptation observed in the cell lines discussed in this review according to the technology used to investigate the function of the corresponding gene.

<p>Summary of the phenotype and adaptation observed in the cell lines discussed in this review according to the technology used to investigate the function of the corresponding gene.</p

Structural and functional characterization of the TgDRE multidomain protein, a DNA repair enzyme from Toxoplasma gondii.

The parasite Toxoplasma gondii expresses a 55 kDa protein or TgDRE that belongs to a novel family of proteins characterized by the presence of three domains, a human splicing factor 45-like motif (SF), a glycine-rich motif (G-patch), and a RNA recognition motif (RRM). The two latter domains are mainly known as RNA-binding domains, and their presence in TgDRE, whose partial DNA repair function was demonstrated, suggests that the protein could also be involved in the RNA metabolism. In this work, we characterized the structure and function of the different domains by using single or multidomain proteins to define their putative role. The SF45-like domain has a helical conformation and is involved in the oligomerization of the protein. The G-patch domain, mainly unstructured on its own as well as in the presence of the SF upstream and RRM downstream domains, is able to bind small RNA oligonucleotides. We also report the structure determination of the RRM domain from the NMR data. It adopts a classical betaalphabetabetaalphabeta topology consisting of a four-stranded beta sheet packed against two alpha helices but does not present the key residues for the RNA interaction. In contrast, our analysis shows that the RRM of TgDRE is not only unable to bind small RNA oligonucleotides but it also shares the protein-protein interaction characteristics with two unusual RRMs of the U2AF heterodimeric splicing factor. The presence of both RNA- and protein-binding domains seems to indicate that TgDRE could also be involved in RNA metabolism

Plasticity between MyoC- and MyoA-Glideosomes: An Example of Functional Compensation in Toxoplasma gondii Invasion

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MyoC-glideosome is not involved in cell division but in invasion.

<p>A. Intracellular growth assay performed on GAP45-iKO and GAP45-iKO/GAP80-KO strains by determining the number of parasites per vacuole after 48 hours ± ATc. Data are represented as mean ± SD. B. Gliding assay performed on poly-L-lysine coated coverslips with GAP45-iKO and GAP45-iKO/GAP80-KO strains after 42 hours ± ATc. C. Ionophore-induced egress assay of GAP45-iKO and GAP45-iKO/GAP80-KO strains was performed by treating the parasites with DMSO or Ca2+-ionophore A23187 for 5 min after 56 hours ± ATc before The results are expressed as a percentage of ruptured vacuoles and represented as mean ± SD. D. Invasiveness of GAP45-iKO and GAP45-iKO/GAP80-KO strains was determined using a two-color immunofluorescence assay performed after 42 hours ± ATc. Intracellular: invaded parasites, extracellular: attached parasites. Data are represented as mean ± SD. The significance of the data was evaluated using a parametric paired t-test and the two-tailed p-value is written on the graph. E. Co-IP performed on metabolically labeled wild type and MyoA-KO parasites using anti-MLC1 antibodies. F. In MyoA-KO, MycMyoC-iKO relocalized to the periphery of the parasites up to the apical basal ring in addition to its basal localization. Two exposures are presented for MycMyoC localization. Scale bars: 2 µm. G. Western-blot of total extract of MycMyoC-iKO and MycMyoC-iKO/MyoA-iKO analyzed using anti-MyoA, anti-MLC1 and anti-Myc antibodies. The loading control was done at the same time with anti-PRF and fluorescent secondary antibodies on the same membrane as MLC1 for the upper panel and as Myc for the lower panel. H. Ionophore-induced egress assay of MyoC-iKO and MyoC-iKO/MyoA-KO strains performed by treating the parasites with DMSO or Ca2+-ionophore A23187 for 5 min after 54 hours ± ATc before The results are expressed as a percentage of ruptured vacuoles and represented as mean ± SD. I. Red/green invasion assay performed after 42 hours ± ATc. Intracellular: invaded parasites, extracellular: attached parasites. Data are represented as mean ± SD. The significance of the data was evaluated using a parametric paired t-test and the two-tailed p-value is written on the graph.</p

Model of redundancy and compensation mechanisms between the MyoA- and MyoC-glidesome of <i>Toxoplasma gondii</i>.

<p>A. Localization and composition of the three glideosomes in <i>T. gondii</i> tachyzoite. B. Illustration of the composition of the basal glideosome according to the component that has been targeted for deletion. C. Illustration of the composition of the glideosomes upon deletion of MyoA.</p