Plasticity between MyoC- and MyoA-glideosomes: an example of functional compensation in Toxoplasma gondii invasion

Abstract

The glideosome is an actomyosin-based machinery that powers motility in Apicomplexa and participates in host cell invasion and egress from infected cells. The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45. In Toxoplasma gondii, GAP45 also contributes to the cohesion of the pellicle, composed of the inner membrane complex (IMC) and the plasma membrane, during motor traction. GAP70 was previously identified as a paralog of GAP45 that is tailored to recruit MyoA at the apical cap in the coccidian subgroup of the Apicomplexa. A third member of this family, GAP80, is demonstrated here to assemble a new glideosome, which recruits the class XIV myosin C (MyoC) at the basal polar ring. MyoC shares the same myosin light chains as MyoA and also interacts with the integral IMC proteins GAP50 and GAP40. Moreover, a central component of this complex, the IMC-associated protein 1 (IAP1), acts as the key determinant for the restricted localization of MyoC to the posterior pole. Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome. Conversely, removal of MyoA leads to the relocalization of MyoC along the pellicle and at the apical cap that accounts for residual invasion. The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival.

Figure 1. A family of GAPs anchored in different sub-compartments of the IMC.

A. Total lysates from Ku80-KO parasites expressing a Ty-tagged endogenous GAP70 or GAP80 (KI: knock-in) analyzed by western blot using anti-Ty antibodies and catalase (CAT) as loading control. B. Localization of KI-GAP70 at the apical cap and KI-GAP80 in a ring-like structure at the basal pole assessed in intracellular parasites using anti-Ty as well as anti-GAP45 or anti-IMC1 antibodies that stain the periphery or the IMC, respectively. Scale bars: 2 µm. C. Immuno-blot of total lysates of RH parasites expressing a second copy of GAP80Ty or GAP70TyCtGAP80 under the control of the tubulin (Tub) promoter. Profilin (PRF) was used as a loading control. The star indicates a degradation product. D. Localization of the second copy of GAP80Ty or GAP70TyCtGAP80 assessed in intracellular parasites using anti-Ty together with anti-MIC4 antibodies that stain the micronemes. Scale bars: 2 µm. E. Total lysates from RH parasites expressing MycGFPCtGAP80 under the control of the tubulin promoter analyzed by western blot using anti-Myc antibodies and CAT as a loading control. F. Localization of MycGFPCtGAP80 at the posterior sub-compartment of the IMC in mature parasites and in growing daughter cells. Scale bars: 2 µm.

Figure 2. Two myosin light chains shared between two motors.

A. The solubility of GAP80 constructs (KI-GAP80Ty and GAP80Ty) was assessed by fractionation after extraction in PBS, PBS/NaCl, PBS/Na₂CO₃ or PBS/Triton X-100. Their distribution in different fractions was assessed by western blot using anti-Ty antibodies and the soluble catalase (CAT) as control for the correct fractionation. B. Parasites stably expressing a second copy of GAP45Ty and GAP80Ty were labeled with [³⁵S]-methionine/cysteine and subjected to co-IP with anti-Ty antibodies. Eluted proteins were visualized by autoradiography. A protein above 130 kDa was additionally found in GAP80Ty elution and identified as MyoC. C. Metabolically labeled parasites endogenously Ty-tagged at the GAP45, GAP80 or MyoC locus have been subjected to co-IP with anti-Ty antibodies. The black circles correspond to the respective bait. The same elution fractions were analyzed by western blot using anti-GAP40 and anti-GAP45 antibodies, respectively. The asterisks indicate the Ig heavy chains that cross-react with the antibodies. D. Autoradiography of labeled parasites stably expressing a second copy of MLC1Ty after co-IP experiment with anti-Ty antibodies. E. Localization of the endogenous MyoC in a ring-like structure at the basal pole of mature parasites and late stage daughter cells using anti-Ty and anti-IMC1 antibodies. Scale bars: 2 µm. F. Autoradiography of labeled parasites stably expressing a Ty-tagged version of the endogenous ELC1 (KI-ELC1-3Ty) after co-IP performed with anti-Ty antibodies. Scale bars: 2 µm. G. Localization of the endogenously tagged ELC1, MLC1 and GAP40 and of loxP-TyMyoA assessed in intracellular parasites using anti-Ty and anti-GAP45 antibodies. Arrowheads point to the basal end of the parasites that are also presented in the magnifications.

Figure 3. Identification of an IMC-associated protein recruiting the MyoC-glideosome to the basal polar ring.

A. Parasites stably expressing MycGFPCtGAP45 and MycGFPCtGAP80 as a second copy were subjected to co-IP with anti-Myc antibodies after metabolic labeling. Eluted proteins were boiled (left panel) or not (right panel) before migration on a SDS-page gel and then visualized by autoradiography. Stars indicate the Myc-tagged proteins. Proteins 1 and 2 are the candidates analyzed by mass spectrometry. B. Total lysates from parasites expressing an endogenous Ty-tagged full-length version of IAP1 (KI-IAP1-3Ty) in a Ku80-KO background or an endogenous Myc-tagged truncated version of IAP1 (KI-Nt-IAP1-3Myc) in the KI-GAP80Ty were analyzed by western blot using respectively anti-Ty and anti-Myc antibodies and the catalase as loading control. C. Localization of KI-IAP1-3Ty at the posterior polar ring and in the last third of the IMC assessed in intracellular parasites using anti-Ty, anti-GAP45 and anti-IMC1 antibodies. Scale bar: 2 µm. D. Schematic representation of the IAP1 constructs used in this study and highlighting the position of the cysteine residues. In red are the cysteines predicted to be palmitoylated by CSS-Palm 3.0 with the highest threshold and in blue are the ones mutated to alanines. E. Co-IP performed with anti-Ty antibodies on parasites expressing KI-IAP1-3Ty and KI-ILP1-3Ty (as control) and revealed by western blot using anti-MLC1 and anti-GAP40 antibodies. Arrows show MLC1 and GAP40, respectively. The asterisks indicate the Ig heavy and light chains that cross-react with the antibodies. F. The truncated version of IAP1 (KI-Nt-IAP1-3Myc) was generated in wild type and KI-GAP80Ty backgrounds. It localizes in the last third of the IMC and led to the relocalization of GAP80Ty in the same area of the IMC. Scale bars: 2 µm. G. A wild type second copy of IAP1 (IAP1-Ty) stably expressed in RH strain localized to the posterior part of the IMC while a second copy of IAP1 mutated on the three first cysteines (AAA-IAP1-Ty) was found in the cytoplasm. Scale bars: 2 µm. H. The solubility of strains expressing IAP1 (KI-IAP1-3Ty, IAP1-Ty and AAA-IAP1-Ty) was assessed by fractionation after extraction in PBS, PBS/NaCl, PBS/Na₂CO₃ or PBS/Triton X-100. Their distribution in different fractions was assessed by western blot using anti-Ty antibodies and the soluble catalase (CAT) was used as control for the correct fractionation.

Figure 4. MyoC is dispensable in tachyzoites.

A. Localization of endogenous MyoC N-terminally Myc-tagged (MycMyoC-iKO) and of endogenous truncated MyoC (KI-MyoC-ΔN&T-3Myc) expressed in KI-GAP80Ty or KI-IAP1-3Ty. Scale bars: 2 µm. B. Total lysates from parasites expressing MycMyoC-iKO and KI-MyoC-ΔN&T-3Myc/KI-GAP80Ty were analyzed by western blot with anti-Myc antibodies. Parasites expressing KI-GAP80Ty were loaded as a control and CAT as a loading control. C. Plaque assays performed with Ku80-KO, KI-GAP80Ty, MyoC-ΔN&T-3Myc/KI-GAP80Ty, MyoC-KO/KI-GAP80Ty cell lines and fixed after 7 days. No defect in the lytic cycle was observed. D. Co-IP experiments performed with anti-Ty antibodies on cell lines metabolically labeled and expressing KI-GAP80Ty in a wild type and in a MyoC-KO background.

Figure 5. The MyoC-glideosome is dispensable in tachyzoites.

A. No growth defect was detected 7 days post-invasion by plaque assays performed with Ku80-KO and GAP80-KO strains. B. In absence of GAP80, endogenous MyoC and IAP1 (KI-MyoC-3Ty and KI-IAP1-3Ty) are still localized to the basal polar ring. Scale bars: 2 µm. C. Co-IP experiments performed with anti-GAP45 antibodies on Ku80-KO and GAP80-KO strains after metabolic labeling with [³⁵S]-methionine/cysteine. D. Plaque assays performed with Ku80-KO and IAP1-KO cell lines and fixed after 7 days. E. Immunofluorescence assays performed on intracellular parasites showing that in absence of IAP1, MyoC and GAP80 (MycMyoC-iKO and KI-GAP80Ty) are not localized to the basal polar ring anymore. Scale bars: 2 µm. F. Western blot analysis of total extract of parasites expressing KI-GAP80Ty in 3 different backgrounds. G. Localization of GAP45 in Ku80-KO, GAP80-KO and IAP1-KO cell lines relatively to GAP40Ty showing that in absence of IAP1 or GAP80, GAP45 staining goes further down to the basal complex as illustrated by the magnifications of the posterior poles and the RGB profile plots determined using ImageJ along the arrow. Scale bars: 2 µm. Little arrows point to the apical pole of the parasites while arrowheads point to the posterior pole.

Figure 6. A non-functional MyoC fails to incorporate into the basal glideosome complex.

A. Localization of MycMyoC-iKO and MycMyoC-K205E-iKO in dividing parasites stained with an IMC marker shows that the mutated MyoC is not incorporated in the mature parasites. Scale bars: 2 µm. B. Western blot analysis of total extract of intracellular parasites expressing MycMyoC-iKO and MycMyoC-K205E-iKO. The mutated MyoC is never detected with anti-Myc antibodies.

Figure 7. GAP80 is able to recruit MLC1-MyoA and to preserve the cohesion of the pellicle.

A. Western blot analysis showing the regulation of the inducible GAP45 (GAP45i), the expression of the complementing Ty-tagged copy of GAP80 (GAP80c) expressed under a tubulin promoter and its stabilization upon addition of ATc for 48 hours. B. Immunofluorescence assay performed on GAP45-iKO/GAP80c strain in absence and in presence of ATc. The arrow indicates a vacuole in which GAP80Ty complements GAP45 deletion and recruits MLC1 at the pellicle while the arrowhead points out a vacuole in which the level of expression of GAP80Ty is not sufficient to rescue GAP45 depletion. Scale bars: 2 µm. C. Plaque assays performed with GAP45-iKO and GAP45-iKO/GAP80c strains treated ± ATc for 7 days. GAP80 restores partially the growth defect of GAP45 depletion. D. Intracellular growth assay performed on GAP45-iKO and GAP45-iKO/GAP80c strains by determining the number of parasites per vacuole after 48 hours ± ATc. Data are represented as mean ± SD. E. Invasion capacity of GAP45-iKO and GAP45-iKO/GAP80c strains was evaluated using a two-color immunofluorescence assay performed after 42 hours ± ATc. Intracellular: invaded parasites, extracellular: attached parasites. Data are represented as mean ± SD. F. Ionophore-induced egress assay of GAP45-iKO and GAP45-iKO/GAP80c strains performed by treating the parasites with DMSO or A23187 for 5 min after 56 hours ± ATc. Results are expressed as a percentage of ruptured vacuoles and represented as mean ± SD. For E and F, the significance of the results was assessed using a parametric paired t-test and the two-tailed p-values are written on the graphs. G. Gliding assay performed with GAP45-iKO and GAP45-iKO/GAP80c strains after 42 hours ± ATc. H. Co-IP carried out with anti-Ty antibodies on GAP45-iKO/GAP80c strain metabolically labeled after 48 hours ± ATc. Upon GAP45 depletion, GAP80 is integrated in MyoA-glideosome and recruits MyoA while the level of bound MyoC remains unchanged.

Figure 8. MyoC-glideosome is not involved in cell division but in invasion.

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.

Figure 9. Model of redundancy and compensation mechanisms between the MyoA- and MyoC-glidesome of Toxoplasma gondii.

A. Localization and composition of the three glideosomes in T. gondii 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.