Figure 5.

The LDVBD domain of TarP and the host protein vinculin are required for focal adhesion resistance to blebbistatin treatment. Top, WT or vinculin-knockout MEFs were infected with C. trachomatis serovar L2. Cells at 20 hpi were mock-treated or treated for 60 min with 10 μm blebbistatin. The cells were then processed for immunofluorescence staining for paxillin (green) and actin (red). Retention or loss of focal adhesions was monitored. Focal adhesions were only resistant to blebbistatin-induced disassembly if the cell was infected and expressing vinculin. Bottom, in a parallel experiment, WT or vinculin-knockout MEFs were transfected for 20 h with the empty vector or LDVBD-mTurquoise2 fusion protein. During the last hour, cells were either mock- or blebbistatin-treated. Cells were processed to visualize paxillin (red), LDVBD (green), and actin (blue; shown in composite images). LDVBD was sufficient to confer resistance to blebbistatin-induced disassembly to focal adhesions. Resistance also required vinculin. Scale bar, 10 μm.