Abstract
Weibel-Palade bodies (WPB) are specialized secretory organelles of endothelial cells that control vascular hemostasis by regulated, Ca2+-dependent exocytosis of the coagulation-promoting von-Willebrand factor. Some proteins of the WPB docking and fusion machinery have been identified but a role of membrane lipids in regulated WPB exocytosis has so far remained elusive. We show here that the plasma membrane phospholipid composition affects Ca2+-dependent WPB exocytosis and von-Willebrand factor release. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] becomes enriched at WPB-plasma membrane contact sites at the time of fusion, most likely downstream of phospholipase D1-mediated production of phosphatidic acid (PA) that activates phosphatidylinositol 4-phosphate (PI4P) 5-kinase γ. Depletion of plasma membrane PI(4,5)P2 or down-regulation of PI4P 5-kinase γ interferes with histamine-evoked and Ca2+-dependent WPB exocytosis and a mutant PI4P 5-kinase γ incapable of binding PA affects WPB exocytosis in a dominant-negative manner. This indicates that a unique PI(4,5)P2-rich environment in the plasma membrane governs WPB fusion possibly by providing interaction sites for WPB-associated docking factors.
Highlights
Vascular homeostasis is delicately balanced to permit unrestricted blood flow and prevent excessive leakage of plasma and blood cells in case of injury
We recorded the behavior of these three phospholipids in the course of histamine-evoked Weibel–Palade bodies (WPB) exocytosis by using multicolor total internal reflection fluorescence (TIRF) microscopy as well as live cell confocal microscopy of primary human endothelial cells (HUVECs) expressing different phospholipidbinding domains as sensors
The cells expressed VWFmRFP as WPB marker to allow for the detection of individual WPB–plasma membrane (PM) fusion events characterized by a collapse of the elongated WPB shape into a spherical object (Erent et al, 2007; Chehab et al, 2017; Mietkowska et al, 2019)
Summary
Vascular homeostasis is delicately balanced to permit unrestricted blood flow and prevent excessive leakage of plasma and blood cells in case of injury. Among the many factors that control this homeostasis is the pro-coagulant glycoprotein von-Willebrand factor (VWF), which recruits platelets to sites of vessel injury and thereby promotes the formation of a platelet plug. The main source of the highly pro-coagulant VWF is endothelial cells which store VWF in unique secretory granules known as Weibel–Palade bodies (WPBs). WPBs are considered lysosome-related organelles that undergo a complex maturation process involving formation at the trans-Golgi network and interactions with the endosomal system (McCormack et al, 2017; Mourik & Eikenboom, 2017). Endothelial stimulation, which occurs in response to blood vessel injury and leads to an intraendothelial Ca2+ and/or cAMP elevation, triggers the exocytosis of mature WPB and the acute release of highly multimeric VWF into the vasculature (Schillemans et al, 2019; Karampini et al, 2020)
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