Figure 4
The outer membrane chimeric protein Intimin-FAST becomes fluorescent upon binding to the non-membrane permeant fluorogen HBRAA-3E. (a) Brightfield (right) and TIRF (left) images of exponentially growing (OD600 = 0.7) E. coli harbouring FAST constructs revealed by the permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens. From top to bottom: cyto-FAST constitutively produced freely in the cytoplasm from the plasmid pZE1R-FAST; Pf3-FAST exposed on the cytoplasmic leaflet of the inner membrane produced from the plasmid pPf3-FAST; Cmi-FAST exposed on the periplasmic leaflet of the inner membrane produced from the plasmid pCmi-FAST; and intimin-FAST exposed at the cell surface produced from pNeae2-FAST, all in presence of IPTG. Cells were deposited on a 1% agarose LB pad containing either 20 µM of HBR-3,5DM or 40 µM of HBRAA-3E with 0.5 mM IPTG when necessary. The background of the fluorescence images has been subtracted. For each construct, fluorescent images obtained with the permeant and non-permeant fluorogens are displayed with the same contrast. Scale bar is 5 µm. (b) The signal of the non-permeant (HBRAA-3E) fluorogen divided by the average of the permeant (HBR-3,5DM) fluorescent signal for the different constructs: cyto-FAST (n = 318 for HBR-3,5DM; n = 55 for HBRAA-3E); Pf3-FAST (n = 211 for HBR-3,5DM; N = 198 for HBRAA-3E); Cmi-FAST (n = 214 for HBR-3,5DM; n = 160 for HBRAA-3) and intimin-FAST (n = 99 for HBR-3,5DM; n = 89 for HBRAA-3E). Error bars represent standard errors. Statistical analysis was performed using unpaired t-tests (***, p value < 0.001; ****, p value < 0.0001).
