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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 ability of the vinculin binding domain to recruit actin is dependent on vinculin. (A)vcl+∕+ and vcl−∕− mouse embryo fibroblasts (MEFs) expressing the Vinculin Binding Domain (TirM-VBD) were infected with Δtir EPEC, and the recruitment of actin monitored. Actin (green) or bacteria (false-colored red) were visualized with phalloidin or DAPI, respectively. The white arrowheads indicate colocalization of actin with Δtir EPEC. Scale bars: 10 μm. (B) Adhered EPEC colocalizing with actin in vcl+∕+ and vcl−∕− MEFs were enumerated 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 170–235 particles was counted per sample. Insets show a magnification of a selected area of the cell. Asterisk indicates statistical significance (One-way ANOVA, Tukey's post-hoc test, P < 0.00001).
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Figure 6: The ability of the vinculin binding domain to recruit actin is dependent on vinculin. (A)vcl+∕+ and vcl−∕− mouse embryo fibroblasts (MEFs) expressing the Vinculin Binding Domain (TirM-VBD) were infected with Δtir EPEC, and the recruitment of actin monitored. Actin (green) or bacteria (false-colored red) were visualized with phalloidin or DAPI, respectively. The white arrowheads indicate colocalization of actin with Δtir EPEC. Scale bars: 10 μm. (B) Adhered EPEC colocalizing with actin in vcl+∕+ and vcl−∕− MEFs were enumerated 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 170–235 particles was counted per sample. Insets show a magnification of a selected area of the cell. Asterisk indicates statistical significance (One-way ANOVA, Tukey's post-hoc test, P < 0.00001).

Mentions: We tested directly the dependence of VBD-mediated actin recruitment on vinculin using vcl−∕− MEFs. Using the same EPEC-based assay in conjunction with TirM-VBD, the transfected fibroblasts were evaluated for actin recruitment at the base of the adhered EPEC. The contrasting cell morphologies of vcl+∕+ and vcl−∕− MEFs were expected and consistent with previous reports. Analysis of the confocal images revealed the essential role of vinculin in VBD-mediated actin recruitment (Figure 6A). Punctate F-actin aggregates could be observed below the adhered EPEC in WT MEFs. In contrast diffuse phalloidin staining were seen in the knockout cells. Quantification of the incidence of actin recruitment confirmed the role of vinculin. The frequencies of actin recruitment were significantly greater (>2.5-fold) relative to the vcl−∕− samples (Figure 6B; p < 0.005; ANOVA and Tukey-Kramer post-hoc test).


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 ability of the vinculin binding domain to recruit actin is dependent on vinculin. (A)vcl+∕+ and vcl−∕− mouse embryo fibroblasts (MEFs) expressing the Vinculin Binding Domain (TirM-VBD) were infected with Δtir EPEC, and the recruitment of actin monitored. Actin (green) or bacteria (false-colored red) were visualized with phalloidin or DAPI, respectively. The white arrowheads indicate colocalization of actin with Δtir EPEC. Scale bars: 10 μm. (B) Adhered EPEC colocalizing with actin in vcl+∕+ and vcl−∕− MEFs were enumerated 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 170–235 particles was counted per sample. Insets show a magnification of a selected area of the cell. Asterisk indicates statistical significance (One-way ANOVA, Tukey's post-hoc test, P < 0.00001).
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Related In: Results  -  Collection

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Figure 6: The ability of the vinculin binding domain to recruit actin is dependent on vinculin. (A)vcl+∕+ and vcl−∕− mouse embryo fibroblasts (MEFs) expressing the Vinculin Binding Domain (TirM-VBD) were infected with Δtir EPEC, and the recruitment of actin monitored. Actin (green) or bacteria (false-colored red) were visualized with phalloidin or DAPI, respectively. The white arrowheads indicate colocalization of actin with Δtir EPEC. Scale bars: 10 μm. (B) Adhered EPEC colocalizing with actin in vcl+∕+ and vcl−∕− MEFs were enumerated 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 170–235 particles was counted per sample. Insets show a magnification of a selected area of the cell. Asterisk indicates statistical significance (One-way ANOVA, Tukey's post-hoc test, P < 0.00001).
Mentions: We tested directly the dependence of VBD-mediated actin recruitment on vinculin using vcl−∕− MEFs. Using the same EPEC-based assay in conjunction with TirM-VBD, the transfected fibroblasts were evaluated for actin recruitment at the base of the adhered EPEC. The contrasting cell morphologies of vcl+∕+ and vcl−∕− MEFs were expected and consistent with previous reports. Analysis of the confocal images revealed the essential role of vinculin in VBD-mediated actin recruitment (Figure 6A). Punctate F-actin aggregates could be observed below the adhered EPEC in WT MEFs. In contrast diffuse phalloidin staining were seen in the knockout cells. Quantification of the incidence of actin recruitment confirmed the role of vinculin. The frequencies of actin recruitment were significantly greater (>2.5-fold) relative to the vcl−∕− samples (Figure 6B; p < 0.005; ANOVA and Tukey-Kramer post-hoc test).

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