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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

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.
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ppat-1004504-g001: 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.

Mentions: A knock-in (KI) strategy in Ku80-KO recipient strain [19], [20] was designed to insert a Ty-tag just upstream of the conserved C-terminal region of GAP70 (KI-GAP70Ty) and GAP80 (KI-GAP80Ty), respectively (Figure S1C). Stable parasite lines confirmed that both genes are expressed in the tachyzoite stage (Figure 1A). GAP80 exhibited the same abnormal migration behavior on SDS-PAGE as previously reported for GAP45 and GAP70 with an apparent molecular weight of 80 kDa whereas the predicted size is 45 kDa. Epitope tagging of GAP70 at the endogenous locus confirmed localization to the apical cap of the parasite previously reported based on expression of a second epitope-tagged copy [14]. In sharp contrast, GAP80 localized exclusively to the basal pole of mature parasites and showed a ring-shaped staining corresponding to the posterior polar ring (Figure 1B). To determine if the C-terminus of GAP80 was sufficient to confer the posterior localization, this domain consisting of the last 85 amino acids (aa) of the protein was either fused to GFP (MycGFPCtGAP80) or exchanged with the corresponding C-terminal domain of GAP70 (GAP70TyCtGAP80) and expressed as a second copy (Figure 1 C, E). As a control, expression of a second copy of GAP80Ty was found mainly targeted to the basal pole, opposite to the apical microneme staining of MIC4, and also slightly at the parasite periphery due to overexpression (Figure 1D). Exchange of the C-terminal domain in GAP70TyCtGAP80 conferred a posterior localization to the otherwise apically localized GAP70 (Figure 1D). MycGFPCtGAP80 also targeted to the basal polar ring, confirming that this C-terminal domain was sufficient to act as a targeting determinant (Figure 1F). As previously observed for the C-terminus of GAP45, the C-terminal domain of GAP80 alone was detectable in the nascent IMC of the daughter cells whereas the full-length proteins were found in the mature pellicle only (Figure 1F). This restriction is likely due to the absence of N-terminal acylation (bioinformatically predicted) in the case of MycGFPCtGAP80, which would anchor GAP80 to the PM prior to its association with the basal pole [14].


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)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231161&req=5

ppat-1004504-g001: 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.
Mentions: A knock-in (KI) strategy in Ku80-KO recipient strain [19], [20] was designed to insert a Ty-tag just upstream of the conserved C-terminal region of GAP70 (KI-GAP70Ty) and GAP80 (KI-GAP80Ty), respectively (Figure S1C). Stable parasite lines confirmed that both genes are expressed in the tachyzoite stage (Figure 1A). GAP80 exhibited the same abnormal migration behavior on SDS-PAGE as previously reported for GAP45 and GAP70 with an apparent molecular weight of 80 kDa whereas the predicted size is 45 kDa. Epitope tagging of GAP70 at the endogenous locus confirmed localization to the apical cap of the parasite previously reported based on expression of a second epitope-tagged copy [14]. In sharp contrast, GAP80 localized exclusively to the basal pole of mature parasites and showed a ring-shaped staining corresponding to the posterior polar ring (Figure 1B). To determine if the C-terminus of GAP80 was sufficient to confer the posterior localization, this domain consisting of the last 85 amino acids (aa) of the protein was either fused to GFP (MycGFPCtGAP80) or exchanged with the corresponding C-terminal domain of GAP70 (GAP70TyCtGAP80) and expressed as a second copy (Figure 1 C, E). As a control, expression of a second copy of GAP80Ty was found mainly targeted to the basal pole, opposite to the apical microneme staining of MIC4, and also slightly at the parasite periphery due to overexpression (Figure 1D). Exchange of the C-terminal domain in GAP70TyCtGAP80 conferred a posterior localization to the otherwise apically localized GAP70 (Figure 1D). MycGFPCtGAP80 also targeted to the basal polar ring, confirming that this C-terminal domain was sufficient to act as a targeting determinant (Figure 1F). As previously observed for the C-terminus of GAP45, the C-terminal domain of GAP80 alone was detectable in the nascent IMC of the daughter cells whereas the full-length proteins were found in the mature pellicle only (Figure 1F). This restriction is likely due to the absence of N-terminal acylation (bioinformatically predicted) in the case of MycGFPCtGAP80, which would anchor GAP80 to the PM prior to its association with the basal pole [14].

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