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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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Chlamydia recruits vinculin to sites of invasion. (A)C. caviae (GPIC) elementary bodies (EBs) were added to Cos7 cells at 4°C, and shifted to 37°C by the addition of pre-warmed media to synchronize invasion. The infection was allowed to proceed for 0, 10, 30, 60, 90, or 120 min at which time the cells were fixed and process for immunofluorescence staining and microscopy. Cells were stained with an anti-vinculin antibody (red) and Chlamydiae were visualized by DAPI (green). Note the increase in the number of co-localization events from 0 to 30 min. Images for the other time points are shown in Figure S1. Scale bars: 5 μm. White arrowheads indicate vinculin colocalizing with GPIC EBs. (B) Colocalization frequency per cell was calculated for each time point up to 120 min post-infection, with at least 50 cells analyzed per time point. There is a rapid increase of vinculin recruitment by 10 min p.i., which was sustained up to 120 min p.i. (C)vcl+∕+ and vcl−∕− cells were infected with C. caviae (GPIC). Infection was allowed to proceed up to 15 min post-temperature shift. Cells were fixed and processed for the invasion assay as detailed in the Materials and Methods Section. GPIC invasion was evaluated in vcl+∕+ and vcl−∕− cells. vcl−∕− cells were less able than wild-type to support GPIC invasion at 5 or 15 min post-infection (p.i.). Data are from a minimum of 150 cells from three independent experiments, and expressed as means ± SD. Asterisk and bar indicates statistical significance between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.008).
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Figure 1: Chlamydia recruits vinculin to sites of invasion. (A)C. caviae (GPIC) elementary bodies (EBs) were added to Cos7 cells at 4°C, and shifted to 37°C by the addition of pre-warmed media to synchronize invasion. The infection was allowed to proceed for 0, 10, 30, 60, 90, or 120 min at which time the cells were fixed and process for immunofluorescence staining and microscopy. Cells were stained with an anti-vinculin antibody (red) and Chlamydiae were visualized by DAPI (green). Note the increase in the number of co-localization events from 0 to 30 min. Images for the other time points are shown in Figure S1. Scale bars: 5 μm. White arrowheads indicate vinculin colocalizing with GPIC EBs. (B) Colocalization frequency per cell was calculated for each time point up to 120 min post-infection, with at least 50 cells analyzed per time point. There is a rapid increase of vinculin recruitment by 10 min p.i., which was sustained up to 120 min p.i. (C)vcl+∕+ and vcl−∕− cells were infected with C. caviae (GPIC). Infection was allowed to proceed up to 15 min post-temperature shift. Cells were fixed and processed for the invasion assay as detailed in the Materials and Methods Section. GPIC invasion was evaluated in vcl+∕+ and vcl−∕− cells. vcl−∕− cells were less able than wild-type to support GPIC invasion at 5 or 15 min post-infection (p.i.). Data are from a minimum of 150 cells from three independent experiments, and expressed as means ± SD. Asterisk and bar indicates statistical significance between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.008).

Mentions: Vinculin has been shown to be a target of a number of microbial pathogens as they take advantage of its ability to mobilize actin. Given previous findings demonstrating the involvement of various focal adhesion components during Chlamydial infection (Coombes and Mahony, 2002; Elwell et al., 2008; Gurumurthy et al., 2010), we sought to determine in greater detail the involvement of vinculin in Chlamydia invasion. Using HeLa cells, vinculin recruitment to the sites of invasion was evaluated. HeLa cells were infected with C. caviae GPIC and vinculin recruitment monitored at 0, 10, 30, 60, 90, and 120 min after synchronization by temperature-shift to 37°C. Cells were immediately fixed with freshly prepared 4% paraformaldehyde at the designated time points. As shown in Figure 1A, vinculin was observed to be strongly concentrated around EBs as early as 10 min post-infection (p.i.). From confocal microscopy images of infected cell monolayers, we quantified the levels of colocalization of EBs to vinculin. The graph in Figure 1B shows recruitment at 10 min p.i., and that the steady state levels remained elevated until at least 120 min p.i.


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)

Chlamydia recruits vinculin to sites of invasion. (A)C. caviae (GPIC) elementary bodies (EBs) were added to Cos7 cells at 4°C, and shifted to 37°C by the addition of pre-warmed media to synchronize invasion. The infection was allowed to proceed for 0, 10, 30, 60, 90, or 120 min at which time the cells were fixed and process for immunofluorescence staining and microscopy. Cells were stained with an anti-vinculin antibody (red) and Chlamydiae were visualized by DAPI (green). Note the increase in the number of co-localization events from 0 to 30 min. Images for the other time points are shown in Figure S1. Scale bars: 5 μm. White arrowheads indicate vinculin colocalizing with GPIC EBs. (B) Colocalization frequency per cell was calculated for each time point up to 120 min post-infection, with at least 50 cells analyzed per time point. There is a rapid increase of vinculin recruitment by 10 min p.i., which was sustained up to 120 min p.i. (C)vcl+∕+ and vcl−∕− cells were infected with C. caviae (GPIC). Infection was allowed to proceed up to 15 min post-temperature shift. Cells were fixed and processed for the invasion assay as detailed in the Materials and Methods Section. GPIC invasion was evaluated in vcl+∕+ and vcl−∕− cells. vcl−∕− cells were less able than wild-type to support GPIC invasion at 5 or 15 min post-infection (p.i.). Data are from a minimum of 150 cells from three independent experiments, and expressed as means ± SD. Asterisk and bar indicates statistical significance between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.008).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Chlamydia recruits vinculin to sites of invasion. (A)C. caviae (GPIC) elementary bodies (EBs) were added to Cos7 cells at 4°C, and shifted to 37°C by the addition of pre-warmed media to synchronize invasion. The infection was allowed to proceed for 0, 10, 30, 60, 90, or 120 min at which time the cells were fixed and process for immunofluorescence staining and microscopy. Cells were stained with an anti-vinculin antibody (red) and Chlamydiae were visualized by DAPI (green). Note the increase in the number of co-localization events from 0 to 30 min. Images for the other time points are shown in Figure S1. Scale bars: 5 μm. White arrowheads indicate vinculin colocalizing with GPIC EBs. (B) Colocalization frequency per cell was calculated for each time point up to 120 min post-infection, with at least 50 cells analyzed per time point. There is a rapid increase of vinculin recruitment by 10 min p.i., which was sustained up to 120 min p.i. (C)vcl+∕+ and vcl−∕− cells were infected with C. caviae (GPIC). Infection was allowed to proceed up to 15 min post-temperature shift. Cells were fixed and processed for the invasion assay as detailed in the Materials and Methods Section. GPIC invasion was evaluated in vcl+∕+ and vcl−∕− cells. vcl−∕− cells were less able than wild-type to support GPIC invasion at 5 or 15 min post-infection (p.i.). Data are from a minimum of 150 cells from three independent experiments, and expressed as means ± SD. Asterisk and bar indicates statistical significance between specific groups (One-way ANOVA, Tukey's post-hoc test, P < 0.008).
Mentions: Vinculin has been shown to be a target of a number of microbial pathogens as they take advantage of its ability to mobilize actin. Given previous findings demonstrating the involvement of various focal adhesion components during Chlamydial infection (Coombes and Mahony, 2002; Elwell et al., 2008; Gurumurthy et al., 2010), we sought to determine in greater detail the involvement of vinculin in Chlamydia invasion. Using HeLa cells, vinculin recruitment to the sites of invasion was evaluated. HeLa cells were infected with C. caviae GPIC and vinculin recruitment monitored at 0, 10, 30, 60, 90, and 120 min after synchronization by temperature-shift to 37°C. Cells were immediately fixed with freshly prepared 4% paraformaldehyde at the designated time points. As shown in Figure 1A, vinculin was observed to be strongly concentrated around EBs as early as 10 min post-infection (p.i.). From confocal microscopy images of infected cell monolayers, we quantified the levels of colocalization of EBs to vinculin. The graph in Figure 1B shows recruitment at 10 min p.i., and that the steady state levels remained elevated until at least 120 min p.i.

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