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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 LD-VBD protein is functional and able to recruit phospho-active or vinculin. (A) Schematic of full length TarP (TarP-FL) or its derivative, the LD-VBD, which represents a combination of the TarP LD domain and the TarP Vinculin Binding Domain (VBD). Please see Figure 4 legend for details. (A,B) Cos7 cells transfected with the plasmid encoding LD-VBD were infected with Δtir EPEC to induce clustering of the proteins. Transfected cells were identified by the capacity to facilitate bacteria attachment. The white arrowheads indicate colocalization of phospho-active FAK (pFAK) or vinculin with adhered Δtir EPEC. pFAK (green) and vinculin (red) were visualized with an anti-FAK (phospho Y397) or an anti-vinculin antibody, respectively. Bacteria [false-colored green (vinculin) or red (pFAK)] were visualized by DAPI. Scale bars: 10 μm. (C,D) Adhered EPEC able to recruit vinculin (C) or pFAK (D) were enumerated and data represented as box and whisker plot. Data compiled from two independent experiments. Plot shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds show means. Roughly 200 particles were counted for both conditions. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). NS; not significant. For comparison, previously quantified data for the LD domain alone or the VBD domain alone with pFAK or vinculin was included.
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Figure 8: The LD-VBD protein is functional and able to recruit phospho-active or vinculin. (A) Schematic of full length TarP (TarP-FL) or its derivative, the LD-VBD, which represents a combination of the TarP LD domain and the TarP Vinculin Binding Domain (VBD). Please see Figure 4 legend for details. (A,B) Cos7 cells transfected with the plasmid encoding LD-VBD were infected with Δtir EPEC to induce clustering of the proteins. Transfected cells were identified by the capacity to facilitate bacteria attachment. The white arrowheads indicate colocalization of phospho-active FAK (pFAK) or vinculin with adhered Δtir EPEC. pFAK (green) and vinculin (red) were visualized with an anti-FAK (phospho Y397) or an anti-vinculin antibody, respectively. Bacteria [false-colored green (vinculin) or red (pFAK)] were visualized by DAPI. Scale bars: 10 μm. (C,D) Adhered EPEC able to recruit vinculin (C) or pFAK (D) were enumerated and data represented as box and whisker plot. Data compiled from two independent experiments. Plot shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds show means. Roughly 200 particles were counted for both conditions. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). NS; not significant. For comparison, previously quantified data for the LD domain alone or the VBD domain alone with pFAK or vinculin was included.

Mentions: In the context of TarP, the VBD represents a further contribution to the growing list of functional domains that subvert the host actin cytoskeleton. One of these is the LD motif, which overlaps with the previously characterized TarP F-actin binding domain (FAB1). This motif mediated a FAK and Arp2/3 complex-dependent actin remodeling (Thwaites et al., 2014). Its close proximity to VBD hinted at a potential interaction between the two domains. Using the EPEC system, the functions of VBD and LD were investigated by expressing the C-terminal 241 amino acid residues of TarP spanning both motifs (TirM-LD-VBD) (Figure 8A). The recruitment of either Tyr397-phosphorylated FAK (pY397-FAK) or vinculin was monitored in parallel with TirM-LD and TirM-VBD (Figure 8B). We reasoned that an additive or synergistic interaction would result in the increased incidence of recruitment of pY397-FAK and vinculin to the clusters of TirM-LD-VBD at the plasma membrane. What we observed was that the incidences of recruitment of vinculin or pY397-FAK by TirM-VBD were similar to those of either the LD or the VBD domain alone (Figures 8C,D), indicating that vinculin recruitment by the VBD motif did not interfere with the recruitment of FAK by the LD domain, and vice versa. There was neither mutual inhibition nor additive/synergistic interactions.


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 LD-VBD protein is functional and able to recruit phospho-active or vinculin. (A) Schematic of full length TarP (TarP-FL) or its derivative, the LD-VBD, which represents a combination of the TarP LD domain and the TarP Vinculin Binding Domain (VBD). Please see Figure 4 legend for details. (A,B) Cos7 cells transfected with the plasmid encoding LD-VBD were infected with Δtir EPEC to induce clustering of the proteins. Transfected cells were identified by the capacity to facilitate bacteria attachment. The white arrowheads indicate colocalization of phospho-active FAK (pFAK) or vinculin with adhered Δtir EPEC. pFAK (green) and vinculin (red) were visualized with an anti-FAK (phospho Y397) or an anti-vinculin antibody, respectively. Bacteria [false-colored green (vinculin) or red (pFAK)] were visualized by DAPI. Scale bars: 10 μm. (C,D) Adhered EPEC able to recruit vinculin (C) or pFAK (D) were enumerated and data represented as box and whisker plot. Data compiled from two independent experiments. Plot shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds show means. Roughly 200 particles were counted for both conditions. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). NS; not significant. For comparison, previously quantified data for the LD domain alone or the VBD domain alone with pFAK or vinculin was included.
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Figure 8: The LD-VBD protein is functional and able to recruit phospho-active or vinculin. (A) Schematic of full length TarP (TarP-FL) or its derivative, the LD-VBD, which represents a combination of the TarP LD domain and the TarP Vinculin Binding Domain (VBD). Please see Figure 4 legend for details. (A,B) Cos7 cells transfected with the plasmid encoding LD-VBD were infected with Δtir EPEC to induce clustering of the proteins. Transfected cells were identified by the capacity to facilitate bacteria attachment. The white arrowheads indicate colocalization of phospho-active FAK (pFAK) or vinculin with adhered Δtir EPEC. pFAK (green) and vinculin (red) were visualized with an anti-FAK (phospho Y397) or an anti-vinculin antibody, respectively. Bacteria [false-colored green (vinculin) or red (pFAK)] were visualized by DAPI. Scale bars: 10 μm. (C,D) Adhered EPEC able to recruit vinculin (C) or pFAK (D) were enumerated and data represented as box and whisker plot. Data compiled from two independent experiments. Plot shows range (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds show means. Roughly 200 particles were counted for both conditions. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). NS; not significant. For comparison, previously quantified data for the LD domain alone or the VBD domain alone with pFAK or vinculin was included.
Mentions: In the context of TarP, the VBD represents a further contribution to the growing list of functional domains that subvert the host actin cytoskeleton. One of these is the LD motif, which overlaps with the previously characterized TarP F-actin binding domain (FAB1). This motif mediated a FAK and Arp2/3 complex-dependent actin remodeling (Thwaites et al., 2014). Its close proximity to VBD hinted at a potential interaction between the two domains. Using the EPEC system, the functions of VBD and LD were investigated by expressing the C-terminal 241 amino acid residues of TarP spanning both motifs (TirM-LD-VBD) (Figure 8A). The recruitment of either Tyr397-phosphorylated FAK (pY397-FAK) or vinculin was monitored in parallel with TirM-LD and TirM-VBD (Figure 8B). We reasoned that an additive or synergistic interaction would result in the increased incidence of recruitment of pY397-FAK and vinculin to the clusters of TirM-LD-VBD at the plasma membrane. What we observed was that the incidences of recruitment of vinculin or pY397-FAK by TirM-VBD were similar to those of either the LD or the VBD domain alone (Figures 8C,D), indicating that vinculin recruitment by the VBD motif did not interfere with the recruitment of FAK by the LD domain, and vice versa. There was neither mutual inhibition nor additive/synergistic interactions.

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