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

Frénal K, Marq JB, Jacot D, Polonais V, Soldati-Favre D - PLoS Pathog. (2014)

Bottom Line: The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45.Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome.The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, CMU, University of Geneva, Geneva, Switzerland.

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.

No MeSH data available.


Related in: MedlinePlus

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 [26] 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.
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ppat-1004504-g003: 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 [26] 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.

Mentions: The C-terminal domain of GAP80 was sufficient to target GFP to the posterior pole and likely also sufficient to recruit the MyoC complex, as it is the case for the MyoA complex with GAP45. Toward the identification of a specific component anchoring the MyoC-glideosome to the basal sub-compartment of the IMC, we completed co-IP experiments with anti-Myc antibodies on parasite strains expressing either MycGFPCtGAP80 or the control MycGFPCtGAP45 (Figure 3A). MycGFPCtGAP80 efficiently immunoprecipitated MyoC, MyoA, MLC1, GAP40 and GAP50. Importantly, two additional components associated with MycGFPCtGAP80 became clearly visible when the samples were not boiled prior to loading on SDS-PAGE suggesting proteins with TMD or strongly associated with membranes [24], [25]. Preparative co-IPs were then performed with MycGFPCtGAP80 and MycGFPCtGAP70 as a control in order to identify the putative anchorage(s) (Figure S3A). Two bands, one below 40 kDa (protein 1) and one above 35 kDa (protein 2), were cut out and 8 and 9 proteins were identified by mass spectrometry, respectively (Table S2). Besides obvious contaminants corresponding to the abundant surface protein SAG1, heat shock and ribosomal proteins, peptides corresponding to MLC1 and GAP50 were also found. More interestingly, peptides corresponding to three hypothetical genes present only in Coccidians and exhibiting a similar cell cycle transcription profile as MyoC were identified and investigated further by epitope tag knock-in at the endogenous locus. The TGME49_283510 product was the only candidate localized to the posterior polar ring and the basal sub-compartment of the IMC and was named IAP1 for IMC-associated protein 1 (Figure 3 B, C and table S3). No transmembrane spanning domain was apparent for IAP1 but instead five cysteine residues were predicted to be palmitoylated with a high probability [26], supporting the strong interaction with the IMC (Figures 3D and S3B). In addition, acylation at multiple sites could explain why IAP1 migrated higher than its expected size, a shift that was even more pronounced when one (Figure S3C) or three acidic Ty-tags (Figure 3B) were added.


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

Frénal K, Marq JB, Jacot D, Polonais V, Soldati-Favre D - PLoS Pathog. (2014)

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 [26] 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.
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Related In: Results  -  Collection

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ppat-1004504-g003: 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 [26] 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.
Mentions: The C-terminal domain of GAP80 was sufficient to target GFP to the posterior pole and likely also sufficient to recruit the MyoC complex, as it is the case for the MyoA complex with GAP45. Toward the identification of a specific component anchoring the MyoC-glideosome to the basal sub-compartment of the IMC, we completed co-IP experiments with anti-Myc antibodies on parasite strains expressing either MycGFPCtGAP80 or the control MycGFPCtGAP45 (Figure 3A). MycGFPCtGAP80 efficiently immunoprecipitated MyoC, MyoA, MLC1, GAP40 and GAP50. Importantly, two additional components associated with MycGFPCtGAP80 became clearly visible when the samples were not boiled prior to loading on SDS-PAGE suggesting proteins with TMD or strongly associated with membranes [24], [25]. Preparative co-IPs were then performed with MycGFPCtGAP80 and MycGFPCtGAP70 as a control in order to identify the putative anchorage(s) (Figure S3A). Two bands, one below 40 kDa (protein 1) and one above 35 kDa (protein 2), were cut out and 8 and 9 proteins were identified by mass spectrometry, respectively (Table S2). Besides obvious contaminants corresponding to the abundant surface protein SAG1, heat shock and ribosomal proteins, peptides corresponding to MLC1 and GAP50 were also found. More interestingly, peptides corresponding to three hypothetical genes present only in Coccidians and exhibiting a similar cell cycle transcription profile as MyoC were identified and investigated further by epitope tag knock-in at the endogenous locus. The TGME49_283510 product was the only candidate localized to the posterior polar ring and the basal sub-compartment of the IMC and was named IAP1 for IMC-associated protein 1 (Figure 3 B, C and table S3). No transmembrane spanning domain was apparent for IAP1 but instead five cysteine residues were predicted to be palmitoylated with a high probability [26], supporting the strong interaction with the IMC (Figures 3D and S3B). In addition, acylation at multiple sites could explain why IAP1 migrated higher than its expected size, a shift that was even more pronounced when one (Figure S3C) or three acidic Ty-tags (Figure 3B) were added.

Bottom Line: The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45.Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome.The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, CMU, University of Geneva, Geneva, Switzerland.

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.

No MeSH data available.


Related in: MedlinePlus