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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

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.
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ppat-1004504-g005: 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.

Mentions: To gain further information about the MyoC-glideosome, a conventional knockout of the GAP80 gene was generated in the Ku80-KO strain (Figure S5E, F). The absence of phenotype by plaque assay indicated that GAP80-KO parasites were able to accomplish their lytic cycle normally (Figure 5A) and indeed the individual steps including intracellular growth and egress were not altered (Figure S5 G, H). Surprisingly, upon deletion of GAP80, neither MyoC nor IAP1 showed an altered localization (Figure 5B). Given the homology between GAP80 and GAP45, it appeared plausible that GAP45 could compensate for the deletion of GAP80. We tested this hypothesis by performing a co-IP using anti-GAP45 antibodies on metabolically labeled wild type parasites and GAP80-KO (Figure 5C). In addition to the MyoA-glideosome components precipitated in the Ku80-KO strain, MyoC was precipitated in GAP80-KO parasites only, confirming that in this mutant strain GAP45 was able to interact with MyoC and hence possibly compensates for the absence of GAP80.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4231161&req=5

ppat-1004504-g005: 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.
Mentions: To gain further information about the MyoC-glideosome, a conventional knockout of the GAP80 gene was generated in the Ku80-KO strain (Figure S5E, F). The absence of phenotype by plaque assay indicated that GAP80-KO parasites were able to accomplish their lytic cycle normally (Figure 5A) and indeed the individual steps including intracellular growth and egress were not altered (Figure S5 G, H). Surprisingly, upon deletion of GAP80, neither MyoC nor IAP1 showed an altered localization (Figure 5B). Given the homology between GAP80 and GAP45, it appeared plausible that GAP45 could compensate for the deletion of GAP80. We tested this hypothesis by performing a co-IP using anti-GAP45 antibodies on metabolically labeled wild type parasites and GAP80-KO (Figure 5C). In addition to the MyoA-glideosome components precipitated in the Ku80-KO strain, MyoC was precipitated in GAP80-KO parasites only, confirming that in this mutant strain GAP45 was able to interact with MyoC and hence possibly compensates for the absence of GAP80.

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