Figure 3. A C‐terminal hGBP1 polybasic motif is required for sustained binding to Shigella flexneri .

• A
  Polymerization of 10 μM hGBP1_F and hGBP1_F ^R584‐586A in the presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm. Absorbance signals were superimposed with nucleotide composition of the same solution analyzed at defined time points, revealing the characteristic first phase of slow polymer nucleation and the second phase of fast polymer growth and cooperative hydrolysis.
• B
  Scanning electron micrographs of live S. flexneri incubated with no protein or with 5 μM of either hGBP1_F, hGBP1_F ^R584‐586A, or hGBP1_F ^R48A (non‐polymerizing mutant) in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces.
• C
  Confocal time‐lapse microscopy frames of formaldehyde‐fixed GFP⁺ S. flexneri following admixture of 10 μM Alexa‐Fluor647‐labeled hGBP1_F or hGBP1_F ^R584‐586A and 2 mM GTP. Binding of hGBP1_F polymers to bacteria at 5 min and enclosure of bacteria with hGBP1_F protein coats after 60 min were quantified. Mean frequencies ± SEM of combined data from at least three independent experiments are shown. Significance was determined by unpaired t‐tests, two‐tailed. ***P ≤ 0.001; ****P ≤ 0.0001.

Data information: All scale bars equal 5 μm.Source data are available online for this figure.