Repetitive N-WASP–Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly
Figure 2
Experimental clustering of the EspFU repeats bypasses the requirement for the Tir C-terminus during actin pedestal formation.
(A) A fusion of membrane-targeted HN-TirFL to EspFU-myc is shown. Treatment of transfected cells with Tir antibodies and S. aureus particles can promote clustering of membrane-localized Tir-EspFU fusions. (B) Murine fibroblast-like cells (FLCs) transfected with plasmids encoding Tir-EspFU fusion constructs comprising the N-terminal domain or truncations of the C-terminal repeats were treated with Tir antibodies and S. aureus, fixed, and stained with HA antibodies to identify both transfected cells and S. aureus (which binds the fluorescent antibodies) and with phalloidin to detect F-actin. (C) Pedestal formation indices were determined by calculating the percentage of transfected cells harboring five or more S. aureus particles generating actin pedestals. Data represent the mean+/−SD from three experiments. The Tir-EspFU R1 construct triggered actin assembly significantly less efficiently than the other truncations (p<0.05).