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Vinculin Interacts with the Chlamydia Effector TarP Via a Tripartite Vinculin Binding Domain to Mediate Actin Recruitment and Assembly at the Plasma Membrane.

Thwaites TR, Pedrosa AT, Peacock TP, Carabeo RA - Front Cell Infect Microbiol (2015)

Bottom Line: The TarP-mediated plasma membrane recruitment of vinculin resulted in the localized recruitment of actin.As further support for the functionality of VBD-vinculin interaction, VBD-mediated actin recruitment required vinculin.Interestingly, while both vinculin and the focal adhesion kinase (FAK) colocalized at the sites of adhesion, the recruitment of one was independent of the other; and the actin recruitment function of the VBD/vinculin signaling axis was independent of the LD/FAK pathway.

View Article: PubMed Central - PubMed

Affiliation: Programme in Microbiology, Institute of Medical Sciences, University of Aberdeen Aberdeen, UK ; Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London London, UK.

ABSTRACT
The mammalian protein vinculin is often a target of bacterial pathogens to subvert locally host cell actin dynamics. In Chlamydia infection, vinculin has been implicated in RNA interference screens, but the molecular basis for vinculin requirement has not been characterized. In this report, we show that vinculin was involved in the actin recruitment and F-actin assembly at the plasma membrane to facilitate invasion. Vinculin was recruited to the plasma membrane via its interaction with a specific tripartite motif within TarP that resembles the vinculin-binding domain (VBD) found in the Shigella invasion factor IpaA. The TarP-mediated plasma membrane recruitment of vinculin resulted in the localized recruitment of actin. In vitro pulldown assays for protein-protein interaction and imaging-based evaluation of recruitment to the plasma membrane demonstrated the essential role of the vinculin-binding site 1 (VBS1), and the dispensability of VBS2 and VBS3. As further support for the functionality of VBD-vinculin interaction, VBD-mediated actin recruitment required vinculin. Interestingly, while both vinculin and the focal adhesion kinase (FAK) colocalized at the sites of adhesion, the recruitment of one was independent of the other; and the actin recruitment function of the VBD/vinculin signaling axis was independent of the LD/FAK pathway.

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C. caviae TarP-mediated vinculin recruitment requires the vinculin binding domain. (A) Schematic indicating the locations of the membrane targeting sequence (yellow box), Ha-tag (brown box), TirM (amino acids 260–395; orange box), actin binding domains (red box), and the LD domains (blue box). The numbers indicate amino acid positions encoded within the C. caviae tarP gene. (B) Cos7 cells transfected with plasmids encoding full length TarP (TirM-TarP FL), progressive TarP deletion derivatives (TirM-TarP1-714 or TirM-TarP-1-639), or the TirM control were infected with Δtir EPEC to induce clustering of the fusion protein. Transfected cells were identified by their ability to bind Δtir EPEC. The white arrowheads indicate colocalization of vinculin (red) with Δtir EPEC (blue). Vinculin was visualized with an anti-vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 5 μm. Refer to Figures 4, 6A for schematic of TarP and its deletion derivatives. (C) Adhered EPEC able to recruit vinculin were enumerated for TirM, TirM-TarP-1-639, TirM-TarP1-714 and TirM-FL-TarP*, and data represented as box and whisker plot. Data compiled from three independent experiments. Plot shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds show means. A range of 360–600 particles was counted. Insets show a magnification of a selected area of the cell. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). NS, not significant.
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Figure 3: C. caviae TarP-mediated vinculin recruitment requires the vinculin binding domain. (A) Schematic indicating the locations of the membrane targeting sequence (yellow box), Ha-tag (brown box), TirM (amino acids 260–395; orange box), actin binding domains (red box), and the LD domains (blue box). The numbers indicate amino acid positions encoded within the C. caviae tarP gene. (B) Cos7 cells transfected with plasmids encoding full length TarP (TirM-TarP FL), progressive TarP deletion derivatives (TirM-TarP1-714 or TirM-TarP-1-639), or the TirM control were infected with Δtir EPEC to induce clustering of the fusion protein. Transfected cells were identified by their ability to bind Δtir EPEC. The white arrowheads indicate colocalization of vinculin (red) with Δtir EPEC (blue). Vinculin was visualized with an anti-vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 5 μm. Refer to Figures 4, 6A for schematic of TarP and its deletion derivatives. (C) Adhered EPEC able to recruit vinculin were enumerated for TirM, TirM-TarP-1-639, TirM-TarP1-714 and TirM-FL-TarP*, and data represented as box and whisker plot. Data compiled from three independent experiments. Plot shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds show means. A range of 360–600 particles was counted. Insets show a magnification of a selected area of the cell. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). NS, not significant.

Mentions: We tested the putative TarP VBD for its ability to recruit vinculin to the plasma membrane using a previously described assay based on the clustering of ectopically expressed and plasma membrane-targeted TarP fused to the extracellular domain of the Tir protein of enteropathogenic Escherichia coli (EPEC) (Thwaites et al., 2014). Briefly, the TarP domain of interest (Figure 3A) was fused to the extracellular and transmembrane domains of Tir. When transiently expressed in host cells, the Tir-TarP fusions (TirM-TarP) were targeted to the plasma membrane where they are clustered by adhered Δtir EPEC to initiate signaling (Campellone et al., 2004; Thwaites et al., 2014). Through deletion analysis of TarP, the heterologous EPEC-based system confirmed the role of VBD (TirM-TarP714−880) in vinculin recruitment. In Figure 3B, vinculin recruitment was induced by full-length TarP (TirM-TarP FL). Removing the C-terminal 166 amino acid residues led to the loss of vinculin recruitment, indicating that this region fully accounted for the vinculin recruitment activity of full-length TarP. Scoring individual EPEC particles for colocalization with vinculin and expressing data as recruitment frequency revealed an increase of three-fold for TarP-FL relative to TirM-TarP1−714 (Figure 3C; p < 0.005; ANOVA and Tukey-Kramer post-hoc test). Taken together, we concluded that TarP itself could recruit vinculin, and we attribute this activity to the putative VBD motif located within the last 166 amino acids.


Vinculin Interacts with the Chlamydia Effector TarP Via a Tripartite Vinculin Binding Domain to Mediate Actin Recruitment and Assembly at the Plasma Membrane.

Thwaites TR, Pedrosa AT, Peacock TP, Carabeo RA - Front Cell Infect Microbiol (2015)

C. caviae TarP-mediated vinculin recruitment requires the vinculin binding domain. (A) Schematic indicating the locations of the membrane targeting sequence (yellow box), Ha-tag (brown box), TirM (amino acids 260–395; orange box), actin binding domains (red box), and the LD domains (blue box). The numbers indicate amino acid positions encoded within the C. caviae tarP gene. (B) Cos7 cells transfected with plasmids encoding full length TarP (TirM-TarP FL), progressive TarP deletion derivatives (TirM-TarP1-714 or TirM-TarP-1-639), or the TirM control were infected with Δtir EPEC to induce clustering of the fusion protein. Transfected cells were identified by their ability to bind Δtir EPEC. The white arrowheads indicate colocalization of vinculin (red) with Δtir EPEC (blue). Vinculin was visualized with an anti-vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 5 μm. Refer to Figures 4, 6A for schematic of TarP and its deletion derivatives. (C) Adhered EPEC able to recruit vinculin were enumerated for TirM, TirM-TarP-1-639, TirM-TarP1-714 and TirM-FL-TarP*, and data represented as box and whisker plot. Data compiled from three independent experiments. Plot shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds show means. A range of 360–600 particles was counted. Insets show a magnification of a selected area of the cell. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). NS, not significant.
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: C. caviae TarP-mediated vinculin recruitment requires the vinculin binding domain. (A) Schematic indicating the locations of the membrane targeting sequence (yellow box), Ha-tag (brown box), TirM (amino acids 260–395; orange box), actin binding domains (red box), and the LD domains (blue box). The numbers indicate amino acid positions encoded within the C. caviae tarP gene. (B) Cos7 cells transfected with plasmids encoding full length TarP (TirM-TarP FL), progressive TarP deletion derivatives (TirM-TarP1-714 or TirM-TarP-1-639), or the TirM control were infected with Δtir EPEC to induce clustering of the fusion protein. Transfected cells were identified by their ability to bind Δtir EPEC. The white arrowheads indicate colocalization of vinculin (red) with Δtir EPEC (blue). Vinculin was visualized with an anti-vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 5 μm. Refer to Figures 4, 6A for schematic of TarP and its deletion derivatives. (C) Adhered EPEC able to recruit vinculin were enumerated for TirM, TirM-TarP-1-639, TirM-TarP1-714 and TirM-FL-TarP*, and data represented as box and whisker plot. Data compiled from three independent experiments. Plot shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds show means. A range of 360–600 particles was counted. Insets show a magnification of a selected area of the cell. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). NS, not significant.
Mentions: We tested the putative TarP VBD for its ability to recruit vinculin to the plasma membrane using a previously described assay based on the clustering of ectopically expressed and plasma membrane-targeted TarP fused to the extracellular domain of the Tir protein of enteropathogenic Escherichia coli (EPEC) (Thwaites et al., 2014). Briefly, the TarP domain of interest (Figure 3A) was fused to the extracellular and transmembrane domains of Tir. When transiently expressed in host cells, the Tir-TarP fusions (TirM-TarP) were targeted to the plasma membrane where they are clustered by adhered Δtir EPEC to initiate signaling (Campellone et al., 2004; Thwaites et al., 2014). Through deletion analysis of TarP, the heterologous EPEC-based system confirmed the role of VBD (TirM-TarP714−880) in vinculin recruitment. In Figure 3B, vinculin recruitment was induced by full-length TarP (TirM-TarP FL). Removing the C-terminal 166 amino acid residues led to the loss of vinculin recruitment, indicating that this region fully accounted for the vinculin recruitment activity of full-length TarP. Scoring individual EPEC particles for colocalization with vinculin and expressing data as recruitment frequency revealed an increase of three-fold for TarP-FL relative to TirM-TarP1−714 (Figure 3C; p < 0.005; ANOVA and Tukey-Kramer post-hoc test). Taken together, we concluded that TarP itself could recruit vinculin, and we attribute this activity to the putative VBD motif located within the last 166 amino acids.

Bottom Line: The TarP-mediated plasma membrane recruitment of vinculin resulted in the localized recruitment of actin.As further support for the functionality of VBD-vinculin interaction, VBD-mediated actin recruitment required vinculin.Interestingly, while both vinculin and the focal adhesion kinase (FAK) colocalized at the sites of adhesion, the recruitment of one was independent of the other; and the actin recruitment function of the VBD/vinculin signaling axis was independent of the LD/FAK pathway.

View Article: PubMed Central - PubMed

Affiliation: Programme in Microbiology, Institute of Medical Sciences, University of Aberdeen Aberdeen, UK ; Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London London, UK.

ABSTRACT
The mammalian protein vinculin is often a target of bacterial pathogens to subvert locally host cell actin dynamics. In Chlamydia infection, vinculin has been implicated in RNA interference screens, but the molecular basis for vinculin requirement has not been characterized. In this report, we show that vinculin was involved in the actin recruitment and F-actin assembly at the plasma membrane to facilitate invasion. Vinculin was recruited to the plasma membrane via its interaction with a specific tripartite motif within TarP that resembles the vinculin-binding domain (VBD) found in the Shigella invasion factor IpaA. The TarP-mediated plasma membrane recruitment of vinculin resulted in the localized recruitment of actin. In vitro pulldown assays for protein-protein interaction and imaging-based evaluation of recruitment to the plasma membrane demonstrated the essential role of the vinculin-binding site 1 (VBS1), and the dispensability of VBS2 and VBS3. As further support for the functionality of VBD-vinculin interaction, VBD-mediated actin recruitment required vinculin. Interestingly, while both vinculin and the focal adhesion kinase (FAK) colocalized at the sites of adhesion, the recruitment of one was independent of the other; and the actin recruitment function of the VBD/vinculin signaling axis was independent of the LD/FAK pathway.

Show MeSH
Related in: MedlinePlus