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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 mediates the recruitment of both vinculin and actin. (A) Cos7 cells transfected with the plasmid encoding the TarP Vinculin Binding Domain (TirM-VBD), the LD domain (TirM-LD), or the control vector TirM were infected with Δtir EPEC to induce clustering of the proteins. Transfected cells were identified by staining for the HA-tag present in the fusion proteins. The white arrowheads indicate colocalization of actin and vinculin with adhered Δtir EPEC. Filamentous actin (green) and vinculin (red) were visualized with phalloidin or an anti-vinculin antibody, respectively. Bacteria (blue) were visualized by DAPI. Scale bars: 10 μm. (B) Adhered EPEC able to recruit actin were enumerated and data represented as box and whisker plot. Data compiled from three independent experiments. Plot shows range, first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 600–730 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).
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Figure 5: The vinculin binding domain mediates the recruitment of both vinculin and actin. (A) Cos7 cells transfected with the plasmid encoding the TarP Vinculin Binding Domain (TirM-VBD), the LD domain (TirM-LD), or the control vector TirM were infected with Δtir EPEC to induce clustering of the proteins. Transfected cells were identified by staining for the HA-tag present in the fusion proteins. The white arrowheads indicate colocalization of actin and vinculin with adhered Δtir EPEC. Filamentous actin (green) and vinculin (red) were visualized with phalloidin or an anti-vinculin antibody, respectively. Bacteria (blue) were visualized by DAPI. Scale bars: 10 μm. (B) Adhered EPEC able to recruit actin were enumerated and data represented as box and whisker plot. Data compiled from three independent experiments. Plot shows range, first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 600–730 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).

Mentions: Having established the role of VBD in vinculin recruitment, we next addressed the functionality of this interaction in regards to actin recruitment and filamentous actin (F-actin) assembly. The ability of the VBD domain to recruit actin was compared to the LD motif, which was previously shown to induce a FAK-dependent signaling to mediate actin recruitment, and thus was the positive control. Using the EPEC-based assay, plasma membrane-localized TirM-VBD, TirM-LD, or TirM was clustered by the adhered EPEC to induce signaling. Recruitment of actin and vinculin was assessed for each sample by staining with phalloidin and anti-vinculin antibody, respectively. As shown in Figure 5A, TirM-VBD and TirM-LD were able to recruit actin, but only the former efficiently localized vinculin underneath the adhered EPEC bacteria. The negative control TirM recruited neither actin nor vinculin. Incidence of actin recruitment was quantified for each sample (Figure 5B). Incidences for both TirM-VBD andTirM-LD were statistically significantly different than that for the negative control. Based on these data, we conclude that the VBD domain is competent in signaling to recruit actin.


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 mediates the recruitment of both vinculin and actin. (A) Cos7 cells transfected with the plasmid encoding the TarP Vinculin Binding Domain (TirM-VBD), the LD domain (TirM-LD), or the control vector TirM were infected with Δtir EPEC to induce clustering of the proteins. Transfected cells were identified by staining for the HA-tag present in the fusion proteins. The white arrowheads indicate colocalization of actin and vinculin with adhered Δtir EPEC. Filamentous actin (green) and vinculin (red) were visualized with phalloidin or an anti-vinculin antibody, respectively. Bacteria (blue) were visualized by DAPI. Scale bars: 10 μm. (B) Adhered EPEC able to recruit actin were enumerated and data represented as box and whisker plot. Data compiled from three independent experiments. Plot shows range, first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 600–730 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).
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Figure 5: The vinculin binding domain mediates the recruitment of both vinculin and actin. (A) Cos7 cells transfected with the plasmid encoding the TarP Vinculin Binding Domain (TirM-VBD), the LD domain (TirM-LD), or the control vector TirM were infected with Δtir EPEC to induce clustering of the proteins. Transfected cells were identified by staining for the HA-tag present in the fusion proteins. The white arrowheads indicate colocalization of actin and vinculin with adhered Δtir EPEC. Filamentous actin (green) and vinculin (red) were visualized with phalloidin or an anti-vinculin antibody, respectively. Bacteria (blue) were visualized by DAPI. Scale bars: 10 μm. (B) Adhered EPEC able to recruit actin were enumerated and data represented as box and whisker plot. Data compiled from three independent experiments. Plot shows range, first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 600–730 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).
Mentions: Having established the role of VBD in vinculin recruitment, we next addressed the functionality of this interaction in regards to actin recruitment and filamentous actin (F-actin) assembly. The ability of the VBD domain to recruit actin was compared to the LD motif, which was previously shown to induce a FAK-dependent signaling to mediate actin recruitment, and thus was the positive control. Using the EPEC-based assay, plasma membrane-localized TirM-VBD, TirM-LD, or TirM was clustered by the adhered EPEC to induce signaling. Recruitment of actin and vinculin was assessed for each sample by staining with phalloidin and anti-vinculin antibody, respectively. As shown in Figure 5A, TirM-VBD and TirM-LD were able to recruit actin, but only the former efficiently localized vinculin underneath the adhered EPEC bacteria. The negative control TirM recruited neither actin nor vinculin. Incidence of actin recruitment was quantified for each sample (Figure 5B). Incidences for both TirM-VBD andTirM-LD were statistically significantly different than that for the negative control. Based on these data, we conclude that the VBD domain is competent in signaling to recruit actin.

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