Abstract

Weibel-Palade bodies (WPBs) are specific cigar-shaped granules that store von Willebrand factor (VWF) for its regulated secretion by endothelial cells. The first steps of the formation of these granules at the trans-Golgi network specifically require VWF aggregation and an external scaffolding complex that contains the adaptator protein complex 1 (AP-1) and clathrin. Discs large 1 (Dlg1) is generally considered to be a modular scaffolding protein implicated in the control of cell polarity in a large variety of cells by specific recruiting of receptors, channels, or signaling proteins to specialized zones of the plasma membrane. We propose here that in endothelial cells, Dlg1, in a complex with AP-1 and clathrin, participates in the biogenesis of WPBs. Supporting data show that Dlg1 colocalizes with microtubules, intermediate filaments, and Golgi markers. Tandem mass spectrometry experiments led to the identification of clathrin as an Dlg1-interacting partner. Interaction was confirmed by in situ proximity ligation assays. Furthermore, AP-1 and VWF immunoprecipitate and colocalize with Dlg1 in the juxtanuclear zone. Finally, Dlg1 depletion by siRNA duplexes disrupts trans-Golgi network morphology and WPB formation. Our results provide the first evidence for an unexpected role of Dlg1 in controlling the formation of specific secretory granules involved in VWF exocytosis in endothelial cells.

Highlights

  • Endothelial cells secrete the hemostatic protein von Willebrand factor (VWF), stored in granules called Weibel-Palade bodies (WPBs)

  • human umbilical vascular endothelial cells (HUVECs) are primary endothelial cells isolated from the human umbilical vein that are well characterized and used extensively in the literature. hCMEC/D3 is an immortalized human brain microvascular endothelial cell line, and these cells are considered as a model of the human blood-brain barrier [21]

  • Discs large 1 (Dlg1) partly colocalizes with GM130, a cis-Golgi marker, and trans-Golgi network protein 2 (TGOLN2), a TGN marker (Fig. 4, I–P). These results suggest that in endothelial cells, Dlg1 is complexed with the adaptator protein complex 1 (AP-1)/clathrin coat of vesicles emerging from the TGN

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Summary

Background

Endothelial cells secrete the hemostatic protein von Willebrand factor (VWF), stored in granules called Weibel-Palade bodies (WPBs). Weibel-Palade bodies (WPBs) are specific cigar-shaped granules that store von Willebrand factor (VWF) for its regulated secretion by endothelial cells. The first steps of the formation of these granules at the trans-Golgi network require VWF aggregation and an external scaffolding complex that contains the adaptator protein complex 1 (AP-1) and clathrin. We propose here that in endothelial cells, Dlg, in a complex with AP-1 and clathrin, participates in the biogenesis of WPBs. Supporting data show that Dlg colocalizes with microtubules, intermediate filaments, and Golgi markers. Our results provide the first evidence for an unexpected role of Dlg in controlling the formation of specific secretory granules involved in VWF exocytosis in endothelial cells. These data provide the first evidence that Dlg, in association with clathrin and AP-1, may control the formation of WPBs at the TGN

EXPERIMENTAL PROCEDURES
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