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. 2014 Sep 1;25(17):2579-91.
doi: 10.1091/mbc.E13-11-0648. Epub 2014 Jul 2.

Calcium-dependent Phosphorylation Alters Class XIVa Myosin Function in the Protozoan Parasite Toxoplasma Gondii

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Calcium-dependent Phosphorylation Alters Class XIVa Myosin Function in the Protozoan Parasite Toxoplasma Gondii

Qing Tang et al. Mol Biol Cell. .
Free PMC article

Abstract

Class XIVa myosins comprise a unique group of myosin motor proteins found in apicomplexan parasites, including those that cause malaria and toxoplasmosis. The founding member of the class XIVa family, Toxoplasma gondii myosin A (TgMyoA), is a monomeric unconventional myosin that functions at the parasite periphery to control gliding motility, host cell invasion, and host cell egress. How the motor activity of TgMyoA is regulated during these critical steps in the parasite's lytic cycle is unknown. We show here that a small-molecule enhancer of T. gondii motility and invasion (compound 130038) causes an increase in parasite intracellular calcium levels, leading to a calcium-dependent increase in TgMyoA phosphorylation. Mutation of the major sites of phosphorylation altered parasite motile behavior upon compound 130038 treatment, and parasites expressing a nonphosphorylatable mutant myosin egressed from host cells more slowly in response to treatment with calcium ionophore. These data demonstrate that TgMyoA undergoes calcium-dependent phosphorylation, which modulates myosin-driven processes in this important human pathogen.

Figures

FIGURE 1:
FIGURE 1:
TgMyoA phosphorylation is enhanced upon treatment with small-molecule enhancers of motility and invasion. (A) RH strain T. gondii tachyzoites metabolically labeled with [32P]orthophosphoric acid were exposed to either 100 μM small-molecule enhancers 153753, 141852, 130038, or 158513 (previously referred to as Enhancers 5, 3, 1, and 6, respectively; Carey et al., 2004) or an equivalent amount of DMSO (vehicle control) for 20 min. The TgMyoA motor complex was then isolated by immunoprecipitation using anti-GAP45 antibody and resolved by SDS–PAGE. Two major phosphorylated bands were visualized by phosphorimaging: TgMyoA (∼93 kDa) and TgGAP45 (∼50 kDa; top). The other 32P–labeled bands in the immunoprecipitate likely include TgGAP40 (∼37 kDa) and TgMLC1 (∼30 kDa), both of which are also know to be multiply phosphorylated (Jacot and Soldati-Favre, 2012). The 32P signals associated with TgMyoA and TgGAP45 were quantified and normalized to the relative amount of TgMyoA protein in the immunoprecipitate (bottom, Sypro-Ruby stain; only the relevant portion of the gel is shown). (B) The normalized TgMyoA and TgGAP45 phosphorylation levels for each treatment are shown as fold change relative to DMSO. *p < 0.05 (one-way analysis of variance [ANOVA]). Experiments were repeated at least twice for each compound; bars, mean value; error bars, SD.
FIGURE 2:
FIGURE 2:
Generation of parasites stably expressing TgMyoA phosphorylation-site mutations. (A) Schematic representation of phosphorylation sites on TgMyoA. The pink vertical lines represent phosphorylation sites identified in this study (S29) or in this study and by others (S20, S21; Nebl et al., 2011; Treeck et al., 2011; www.toxodb.org). The different domains of TgMyoA are shown in light purple (N-terminal domain; amino acids [aa] 1–93, uncharacterized function), green (motor domain; aa 94–774), yellow (tail domain; aa 775–831), and blue (IQ motif; aa 808–814). (B) TgMyoA mutations generated in this study and the abbreviated names of the strains expressing them. (C) N-terminally FLAG-tagged TgMyoA (wild type or mutant) was expressed in TgMyoA KO parasites under the control of the endogenous TgMyoA promoter. Lysates from each of the strains were analyzed by SDS–PAGE and Western blotting with anti-TgMyoA (green); anti-TgGRA8 (red) was used as a loading control. The TgMyoA WT6A and TgMyoA SAAA6A samples were from a twice-bleomycin-selected population (>98% FLAG-positive parasites by immunofluorescence), whereas all other parasite lines were clonal. (D) The cellular distributions of the expressed proteins were analyzed by immunofluorescence microscopy using mouse anti-FLAG antibody (red); rabbit anti-TgIMC1 was used to label the inner membrane complex (green). Scale bars, 10 μm.
FIGURE 3:
FIGURE 3:
The S21A and SAAA mutations reduce basal and 1300038-enhanced TgMyoA phosphorylation. (A) Tachyzoites of the indicated strains were metabolically labeled with [32P]orthophosphoric acid and exposed to either 100 μM 130038 (+) or DMSO (vehicle; –) for 20 min. The TgMyoA motor complex was then isolated by immunoprecipitation using anti-TgGAP45 antibody and resolved by SDS–PAGE; the 32P signal associated with TgMyoA was analyzed as in Figure 1, except that the relative amount of TgMyoA present in the immunoprecipitate was determined by Oriole (bottom) rather than Sypro Ruby staining. (B) TgMyoA phosphorylation under each condition shown as the intensity of the TgMyoA-associated 32P signal, normalized to the amount TgMyoA protein present in the immunoprecipitate. Values are plotted relative to DMSO-treated TgMyoA WT, which was arbitrarily set to 100. Experiments were repeated twice; error bars, SD. **p < 0.01 and ****p < 0.0001 (two-way ANOVA analysis and Bonferroni multiple comparisons test).
FIGURE 4:
FIGURE 4:
Enhancement of TgMyoA phosphorylation by 130038 is calcium dependent. (A) RH strain tachyzoites metabolically labeled with [32P]orthophosphoric acid were pretreated with BAPTA-AM (20 μM) or vehicle (DMSO) for 20 min. Compound 130038 was then added where indicated to a final concentration of 100 μM, and samples were incubated for an additional 15 min. The TgMyoA motor complex was then isolated by immunoprecipitation using anti-TgGAP45 antibody and resolved by SDS–PAGE; the 32P signal associated with TgMyoA was analyzed as in Figure 3. Images were cropped from selected lanes from the same phosphorimager scan/stained gel; all lanes were processed identically. (B) Normalized TgMyoA phosphorylation level for each treatment in A shown as fold change relative to DMSO, no 130038 treatment. A similar analysis showed no significant change in TgGAP45 phosphorylation after enhancer treatment. (C, D) RH strain tachyzoites were metabolically labeled with [32P]orthophosphoric acid and exposed to DMSO (vehicle control), A23187 (2 μM), or BAPTA-AM (20 μM) for 20 min, immunoprecipitated, and processed as in A and B. Images were cropped from selected lanes from the same phosphorimager scan/stained gel; all lanes were processed identically. All experiments were repeated twice; error bars, SD. ***p < 0.001 (one-way ANOVA).
FIGURE 5:
FIGURE 5:
The S21A and SAAA mutations block calcium-induced phosphorylation of TgMyoA. (A) Tachyzoites of the indicated strains were metabolically labeled with [32P]orthophosphoric acid and exposed to either 2 μM A23187 or DMSO (vehicle) for 15 min. The TgMyoA motor complex was then isolated by immunoprecipitation using anti-TgGAP45 antibody and resolved by SDS–PAGE; the 32P signal associated with TgMyoA was analyzed as in Figure 3. (B) TgMyoA phosphorylation under each condition shown as normalized intensity of the TgMyoA-associated 32P signal, as described in Figure 3. Experiments were repeated twice, error bars, SD. ****p < 0.0001 (two-way ANOVA analysis and Bonferroni multiple comparisons test).
FIGURE 6:
FIGURE 6:
Compound 130038 stimulates Ca2+ release into the cytosol of T. gondii tachyzoites. (A) The addition of various concentrations of 130038 to Fura 2-AM–labeled parasites at 100 s demonstrates that 130038 stimulates a dose-dependent increase in cytosolic Ca2+. The extracellular buffer was Ringer containing 100 μM ethylene glycol tetraacetic acid. The control trace shows the effect of adding DMSO at the maximum volume used (5 μl or 0.2%). (B) Experimental conditions were similar to A, but the extracellular buffer contained 1 mM Ca2+.
FIGURE 7:
FIGURE 7:
The SEDD phosphomimetic mutations do not enhance phosphorylation of other sites on TgMyoA. (A) Tachyzoites of the indicated strains were metabolically labeled with [32P]orthophosphoric acid and exposed to DMSO (vehicle control), 100 μM 130038, or 2 μM A23187 for 20 min. The TgMyoA motor complex was then isolated by immunoprecipitation using anti-TgGAP45 antibody and resolved by SDS–PAGE; the 32P signal associated with TgMyoA was analyzed as in Figure 3. (B) TgMyoA phosphorylation under each condition shown as normalized intensity of the TgMyoA-associated 32P signal, as described in Figure 3. Experiments were repeated twice; error bars, SD. Statistically significant differences were observed between the two strains after treatment with either 130038 or A23187 (****p < 0.0001; two-way ANOVA analysis and Bonferroni multiple comparisons test); no significant difference was observed in TgMyoA SEDD parasites between treatments.
FIGURE 8:
FIGURE 8:
The effect of TgMyoA phosphorylation site mutations on parasite invasion and motility. (A) The ability of TgMyoA KO and TgMyoA WT, TgMyoA S21A, and TgMyoA SAAA complemented strains to invade host cells in the presence or absence of 100 μM 130038 was determined using the laser scanning cytometry–based invasion assay. Mean number of intracellular parasites per scan area for each condition. Experiments were repeated three times; error bars, SD. *p < 0.05 (two-way ANOVA analysis and Bonferroni multiple comparisons test). (B) Quantification of parasite motility by 2D live videomicroscopy. Parasite motile behavior is plotted as the mean percentage of total parasites in the observed fields exhibiting twirling, circular, or helical motility. Including motile and nonmotile parasites, n = 350–650 parasites were analyzed per treatment for each parasite line; the experiment was performed twice; error bars, SD. Two-way ANOVA was performed on each type of motility; to preserve statistical power, an uncorrected Fisher least significant difference test was performed on each type of motility for multiple comparisons between strains and treatments. *p < 0.05.
FIGURE 9:
FIGURE 9:
Parasites expressing the SAAA TgMyoA mutation show a delay in calcium-induced host cell egress. HFF cells were infected with TgMyoA WT, TgMyoA SAAA, or TgMyoA SEDD parasites and exposed 36–48 h postinfection to 0.1 μM calcium ionophore A23187 for the times indicated. The number of vacuoles from which parasites had egressed was determined by immunofluorescence microscopy and is plotted as the mean percentage of the total number of vacuoles (egressed plus intact) observed. Experiments were repeated three times; error bars, SD. Statistically significant differences were observed between A23187-treated TgMyoA WT and TgMyoA SAAA parasites at the time points indicated. ***p < 0.001, ****p < 0.0001 (two-way ANOVA with Bonferroni multiple comparison test).

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