Figure 6. Endothelial pore confinement is driven by actomyosin contractility.

(a) Immunofluorescence analyses of MLC phosphorylation during neutrophil transmigration. Open and filled arrows indicate Lifeact-mCherry (red) and MLC phosphorylation (green) localization, respectively, during neutrophil transmigration under physiological flow conditions (0.8 dyne per cm²). Asterisk indicates extravasating leukocyte in DIC. Scale bar, 10 μm. (b) Quantification of F-actin distribution in endothelial pores surrounding transmigrating neutrophils and monocytes. Maximum intensity projection of F-actin in the endothelial pore was used to quantify F-actin distribution surrounding transmigrating leukocytes. Distribution asymmetry is defined by the ratio of region of interest ROI-1 and ROI-2 corrected for background. Scale bar, 5 μm. (c) Confocal imaging of F-actin dynamics during leukocyte diapedesis in retina vasculature of Lifeact-EGFP C57BL6 mice. Filled arrows indicate the vasculature of mice retina, highly expressing Lifeact-GFP. Zoom of ROI, open arrows indicate the Lifeact-EGFP (green)-positive endothelial pore, filled arrows indicate transmigrating neutrophil. Scale bar, 5 μm. (d) Quantification of endothelial pore size in retina vasculature. (e) Confocal imaging of PECAM-1 in cremasteric venules during TNF-α and IL-1β induced neutrophil recruitment. Open arrows indicate PECAM-1 positive endothelial pores that surround extravasating neutrophils (filled arrows). Scale bar, 20 μm. (f) IF analyses of MLC phosphorylation during neutrophil transmigration into the cremaster of C57BL6 mice. Filled and open arrows indicate phospo-MLC and PECAM-1 localization to endothelial pores, respectively. Scale bar, 5 μm. (g) Quantification of pMLC and PECAM-1 localization in endothelial pores. We quantified MLC phosphorylation defined as distribution asymmetry. The distribution asymmetry uses the intensity of one ROI versus another ROI as indicated in b. Because MLC may occur at different heights within the pore we used max projection for this analysis. Data are representative of three independent experiments (a–g) with >12 transmigration events per group (error bars (b,d,g) s.e.m).