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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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The vinculin binding domain of TarP is functional and able to recruit vinculin. (A) Schematic of full length C. caviae TarP deleted of the proline rich domain (TarP FL*) and the TarP Vinculin Binding Domain (VBD; TirM-VBD) indicating the locations of the N-terminal membrane targeting sequence (yellow box), Ha-tag (brown box), TirM (amino acids 260–395; orange box), actin binding domains (red box), the LD domains (blue box), Vinculin Binding Site 3 (VBS; brown box), VBS2 (light orange box), and C-terminal VBS1 (yellow box). The numbers indicate amino acid positions encoded within the C. caviae TarP gene. *Denotes TarP derivative deleted for the PRD. (B) Cos7 cells transfected with plasmids encoding the VBD (TirM-VBD), the LD domain (TirM-LD), 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. Heat map of the vinculin channel highlights the intensity of vinculin recruited to sites of bacterial adherence. The white arrowheads indicate colocalization of vinculin (red) with Δtir EPEC (false-colored green). Vinculin was visualized with an anti-vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 10 μm. Heat map representation (red, high; violet, low) was generated using imageJ. (C) Adhered EPEC able to recruit vinculin were enumerated for TirM, TirM-LD and TirM_VBD, 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 310–680 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 4: The vinculin binding domain of TarP is functional and able to recruit vinculin. (A) Schematic of full length C. caviae TarP deleted of the proline rich domain (TarP FL*) and the TarP Vinculin Binding Domain (VBD; TirM-VBD) indicating the locations of the N-terminal membrane targeting sequence (yellow box), Ha-tag (brown box), TirM (amino acids 260–395; orange box), actin binding domains (red box), the LD domains (blue box), Vinculin Binding Site 3 (VBS; brown box), VBS2 (light orange box), and C-terminal VBS1 (yellow box). The numbers indicate amino acid positions encoded within the C. caviae TarP gene. *Denotes TarP derivative deleted for the PRD. (B) Cos7 cells transfected with plasmids encoding the VBD (TirM-VBD), the LD domain (TirM-LD), 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. Heat map of the vinculin channel highlights the intensity of vinculin recruited to sites of bacterial adherence. The white arrowheads indicate colocalization of vinculin (red) with Δtir EPEC (false-colored green). Vinculin was visualized with an anti-vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 10 μm. Heat map representation (red, high; violet, low) was generated using imageJ. (C) Adhered EPEC able to recruit vinculin were enumerated for TirM, TirM-LD and TirM_VBD, 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 310–680 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: 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.


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)

The vinculin binding domain of TarP is functional and able to recruit vinculin. (A) Schematic of full length C. caviae TarP deleted of the proline rich domain (TarP FL*) and the TarP Vinculin Binding Domain (VBD; TirM-VBD) indicating the locations of the N-terminal membrane targeting sequence (yellow box), Ha-tag (brown box), TirM (amino acids 260–395; orange box), actin binding domains (red box), the LD domains (blue box), Vinculin Binding Site 3 (VBS; brown box), VBS2 (light orange box), and C-terminal VBS1 (yellow box). The numbers indicate amino acid positions encoded within the C. caviae TarP gene. *Denotes TarP derivative deleted for the PRD. (B) Cos7 cells transfected with plasmids encoding the VBD (TirM-VBD), the LD domain (TirM-LD), 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. Heat map of the vinculin channel highlights the intensity of vinculin recruited to sites of bacterial adherence. The white arrowheads indicate colocalization of vinculin (red) with Δtir EPEC (false-colored green). Vinculin was visualized with an anti-vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 10 μm. Heat map representation (red, high; violet, low) was generated using imageJ. (C) Adhered EPEC able to recruit vinculin were enumerated for TirM, TirM-LD and TirM_VBD, 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 310–680 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 4: The vinculin binding domain of TarP is functional and able to recruit vinculin. (A) Schematic of full length C. caviae TarP deleted of the proline rich domain (TarP FL*) and the TarP Vinculin Binding Domain (VBD; TirM-VBD) indicating the locations of the N-terminal membrane targeting sequence (yellow box), Ha-tag (brown box), TirM (amino acids 260–395; orange box), actin binding domains (red box), the LD domains (blue box), Vinculin Binding Site 3 (VBS; brown box), VBS2 (light orange box), and C-terminal VBS1 (yellow box). The numbers indicate amino acid positions encoded within the C. caviae TarP gene. *Denotes TarP derivative deleted for the PRD. (B) Cos7 cells transfected with plasmids encoding the VBD (TirM-VBD), the LD domain (TirM-LD), 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. Heat map of the vinculin channel highlights the intensity of vinculin recruited to sites of bacterial adherence. The white arrowheads indicate colocalization of vinculin (red) with Δtir EPEC (false-colored green). Vinculin was visualized with an anti-vinculin antibody. Bacteria were visualized by DAPI staining. Scale bars: 10 μm. Heat map representation (red, high; violet, low) was generated using imageJ. (C) Adhered EPEC able to recruit vinculin were enumerated for TirM, TirM-LD and TirM_VBD, 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 310–680 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: 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.

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