Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules

Abstract

Endothelial exocytosis of Weibel–Palade body (WPB) is one of the first lines of defence against vascular injury. However, the mechanisms that control WPB exocytosis in the final stages (including the docking, priming and fusion of granules) are poorly understood. Here we show that the focal adhesion protein zyxin is crucial in this process. Zyxin downregulation inhibits the secretion of von Willebrand factor (VWF), the most abundant cargo in WPBs, from human primary endothelial cells (ECs) induced by cAMP agonists. Zyxin-deficient mice exhibit impaired epinephrine-stimulated VWF release, prolonged bleeding time and thrombosis, largely due to defective endothelial secretion of VWF. Using live-cell super-resolution microscopy, we visualize previously unappreciated reorganization of pre-existing actin filaments around WPBs before fusion, dependent on zyxin and an interaction with the actin crosslinker α-actinin. Our findings identify zyxin as a physiological regulator of endothelial exocytosis through reorganizing local actin network in the final stage of exocytosis.

Tuning the endothelial response: differential release of exocytic cargos from Weibel‐Palade bodies

“…C,D, and Figure S3C–F). Although thrombin relies minimally on the actin ring for release of VWF, PMA, VEGF, histamine/adrenalin/IBMX and forskolin are strong ring recruiters (correlating with our and others’ findings ). We also find that actin ring recruitment can be enhanced via addition of some, but not all, calcium or cAMP‐raising agents (Fig.…”

“…Our research complements recent research confirming that VWF release is boosted by an actomyosin ring [25,26]. However, there are differences in the findings.…”

“…We and others demonstrated that VWF release is boosted by a contractile actomyosin ring forming around the fused WPB to squeeze out content [9,25,26], but whether this boost affects platelet and leukocyte recruitment to endothelial cells was undetermined. We first analyzed the effect of actomyosin ring inhibition on VWF string formation ( Fig.…”

“…We also differ on whether WPB localization is generally affected by myosin II inhibition [9,26]. The different conclusions might reflect which cell surface was imaged (apical vs. basal) or spinning disk vs. custom microscopy [9,25]. A role for some actin nucleation remains a possibility.…”

“…Because PKCα can be activated directly by both DAG and calcium or indirectly via cAMP‐dependent agonists and EPAC (exchange proteins directly activated by cAMP isoform) , PKC activation could feasibly occur downstream of any of the ring‐recruiting agonists that we, and others, have identified. The cAMP‐raising secretagogues forskolin and adrenalin have previously been identified by others as stimulating actin ring recruitment. Here we find that addition of adrenalin/IBMX to the calcium‐dependent secretagogue histamine significantly enhances ring recruitment, suggesting that activation of cAMP may be an important route to ring recruitment.…”