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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 induces robust actin recruitment. (A) 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 actin with adhered Δtir EPEC. Actin (green) and bacteria (false-colored red) were visualized with phalloidin or DAPI, respectively. 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 (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 300–680 particles were counted. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). For comparison, previously quantified data for the LD domain alone or the VBD domain alone actin was included.
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Figure 9: The LD-VBD induces robust actin recruitment. (A) 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 actin with adhered Δtir EPEC. Actin (green) and bacteria (false-colored red) were visualized with phalloidin or DAPI, respectively. 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 (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 300–680 particles were counted. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). For comparison, previously quantified data for the LD domain alone or the VBD domain alone actin was included.

Mentions: The LD, VBD, and LD-VBD domains were evaluated for actin recruitment using the EPEC assay by staining the samples with phalloidin and DAPI to visualize filamentous actin and adhered EPEC bacteria on the cell surface, respectively (Figure 9A). Incidences of recruitment were monitored in a single-blind experiment, and data was expressed as a box and whisker plot (Figure 9B). TirM-LD-VBD was able to recruit actin at a frequency similar to that obtained for TirM-VBD. The value obtained for TirM-LD was consistent with our previous report (Thwaites et al., 2014). Based on the data, the FAK/LD and VBD/vinculin pathways do not exhibit an additive or synergistic interactions. It was not feasible to distinguish the relative contributions of each to the LD-VBD-mediated actin recruitment, and we could not exclude the possibility that signaling from the VBD inhibited that from the LD domain. We deem this unlikely, as FAK recruitment by the LD motif was not inhibited when VBD and vinculin were present.


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 induces robust actin recruitment. (A) 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 actin with adhered Δtir EPEC. Actin (green) and bacteria (false-colored red) were visualized with phalloidin or DAPI, respectively. 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 (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 300–680 particles were counted. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). For comparison, previously quantified data for the LD domain alone or the VBD domain alone actin was included.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4663276&req=5

Figure 9: The LD-VBD induces robust actin recruitment. (A) 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 actin with adhered Δtir EPEC. Actin (green) and bacteria (false-colored red) were visualized with phalloidin or DAPI, respectively. 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 (statistical outliers excluded), first and third quartiles, and overall median (horizontal line). Diamonds indicate means. A range of 300–680 particles were counted. The asterisk and bars indicate significance difference between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.00001). For comparison, previously quantified data for the LD domain alone or the VBD domain alone actin was included.
Mentions: The LD, VBD, and LD-VBD domains were evaluated for actin recruitment using the EPEC assay by staining the samples with phalloidin and DAPI to visualize filamentous actin and adhered EPEC bacteria on the cell surface, respectively (Figure 9A). Incidences of recruitment were monitored in a single-blind experiment, and data was expressed as a box and whisker plot (Figure 9B). TirM-LD-VBD was able to recruit actin at a frequency similar to that obtained for TirM-VBD. The value obtained for TirM-LD was consistent with our previous report (Thwaites et al., 2014). Based on the data, the FAK/LD and VBD/vinculin pathways do not exhibit an additive or synergistic interactions. It was not feasible to distinguish the relative contributions of each to the LD-VBD-mediated actin recruitment, and we could not exclude the possibility that signaling from the VBD inhibited that from the LD domain. We deem this unlikely, as FAK recruitment by the LD motif was not inhibited when VBD and vinculin were present.

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