Septin rings are recruited to WPBs post fusion in an actin-independent but PAK2-dependent process. (A) To form higher-order structures such as rings, septin (SEPT) monomers from different subfamilies (eg, SEPT2/6/7/9) must form hetero-oligomers (6mers or 8mers). (B) SEPT7 is recruited to WPBs following stimulation. IF analyses of SEPT7 (green), VWF (blue), and actin (magenta) in HUVEC cultures stimulated with the presence or absence 100 ng/mL PMA and/or 1 μM CCE. Scale bar, 10 μm. (C) Schematic representation of the approach used to image septins in live endothelial cells. GFP-tagged SEPT6 interacts with endogenous SEPT7 upon ring formation. (D) GFP pull-down of control GFP and eGFP-SEPT6−transfected cells detects the association of the eGFP-SEPT6 transgene product with endogenous SEPT2 and SEPT7. Beta tubulin was absent in both eGFP control and eGFP-SEPT6 pull-down samples. ∗Cross-reactivity. (E) Confocal microscopy of fixed HUVECs transfected with P.sel.lum.mCherry (magenta) and eGFP-SEPT6 (green) and probed for SEPT7 (red) by IF. Images show that eGFP-SEPT6 is incorporated into rings that are positive for SEPT7 staining. Scale bars, 10 μm. (F) SEPT9 is localized to fused WPBs following stimulation with PMA. (G) Live cell imaging of PMA (100 ng/mL)−stimulated HUVECs indicates that SEPT6-eGFP rings form post fusion. Brightness and contrast increased for clarity. †WPB fusion. ∗Septin ring formation; 0.5- to 6-μm Z stacks acquired every 5 seconds for 10 minutes. Scale bars, 1 μm. (H) HUVECs were transfected with LUC control (left panel) or PAK2-targeted siRNA (right panel) and probed for SEPT7 (green), VWF (blue), and actin (magenta). Scale bar, 10 μm. (I) Quantification of the number of SEPT7 associated with VWF as a proportion of the total number of rounded (fused) WPBs. A total of 15 images per condition from three independent experiments (ratio paired t test). ∗∗∗P < .005.