Fig. 2.

Exocytosis is up-regulated in Listeria protrusions. (A) Results in subconfluent Caco-2 BBE1 cells. Cells expressing VAMP3-pHluorin were infected with WT or ∆inlC mutant strains of Listeria for 1.5 h in the absence of gentamicin and another 4.5 h in the presence of gentamicin, followed by fixation and labeling of exofacial VAMP3-pHluorin [exo V3-pH (red), F-actin (green), or DNA (white)]. (i) Representative confocal microscopy images of bacterial protrusions are presented. Protrusions are indicated by arrows, and regions around protrusions are expanded in panels on the Right. White objects at the end of protrusions are bacteria. (Scale bars, 5 µm.) (ii) FE data for exofacial VAMP3-pHluorin (exo V3) in bacterial protrusions are presented. Each dot represents an FE measurement. Data are mean ± SD of 165 or 166 FE values for WT or ∆inlC Listeria strains, respectively. *P < 0.0001. (B) Results in confluent Caco-2 BBE1 cells. Caco-2 BBE1 cells stably expressing VAMP3-pHluorin were mixed with normal Caco-2 BBE1 cells at a ratio of 1:3 and grown to confluency. Cells were infected with WT or ∆inlC strains of Listeria for 1.5 h in the absence of gentamicin and another 4.5 h in the presence of gentamicin, followed by labeling of exofacial VAMP3-pHluorin [exo V3-pH (red), F-actin (green), and DNA (white)]. Intrinsic GFP fluorescence [total V3-pH (blue)] was used to identify protrusions from cells expressing VAMP3-pHluorin that project into surrounding cells lacking VAMP3-pHluorin. (i) Representative confocal microscopy images are shown. Protrusions are indicated with arrows on the Left. Regions around protrusions are enlarged in panels on the Right. White objects (labeled for DNA) are bacteria. (Scale bars, 5 µm.) (ii) FE data for exofacial VAMP3-pHluorin (exo V3) in protrusions are displayed. Each dot indicates an FE measurement. Data are mean ± SD of 151 or 173 FE values for WT or ∆inlC Listeria strains, respectively. *P < 0.0001.