Endothelial β-PIX (ARHGEF7) drives exocytosis through enabling the dynamic reorganisation of the cytoskeleton

Abstract

Endothelial cells rapidly respond to blood vessel injury or infection by releasing haemostatic and inflammatory proteins from their secretory organelles called Weibel-Palade Bodies (WPBs). Upon stimulation, WPBs traffic to the cell surface to secrete cargo such as von Willebrand factor (VWF), essential for platelet recruitment, and P-selectin, which facilitates the capture and subsequent rolling of immune cells. While this process is critical, many aspects of WPB trafficking and exocytosis remain unclear--particularly how release sites are selected across the cell surface and how fusion is directed to either the apical (lumen-facing) or basolateral (basement membrane-facing) side. These decisions directly impact the biological consequences and effectiveness of the secreted proteins. Using live-cell imaging, fibronectin micropatterning, loss-of-function and biochemical assays, we identified trafficking and release at hot spots near focal adhesions (FAs). We also discovered that the FA-resident guanine nucleotide exchange factor (GEF), {beta}-PIX (ARHGEF7), regulates VWF secretion. {beta}-PIX is known to activate Rho GTPases (Cdc42/Rac), triggering downstream p21-activated kinase 2 (PAK2) signalling and cytoskeletal remodelling. Here, depletion of {beta}-PIX impaired VWF secretion and delayed its release by perturbing cytoskeletal reorganisation. Knockdown and rescue experiments using truncated mutants further revealed which domains of {beta}-PIX are necessary for exocytosis and cytoskeletal reorganisation. This is the first demonstration of {beta}-PIXs role in VWF secretion from endothelial cells and our data provides new insights into spatial targeting of WPB exocytosis. Such targeting may be essential for guiding leukocyte transmigration or platelet binding, thereby maintaining vascular integrity. SchematicEndothelial secretory organelles, known as Weibel-Palade bodies (WPBs), store von Willebrand factor (VWF), and their regulated exocytosis is critical for blood clotting and immune responses. This process can be stimulated experimentally by phorbol esters as well as physiological stimuli such as adrenaline and histamine. Cytoskeletal remodelling is required for both pre- and post-fusion events in this secretory pathway. Here, we present evidence supporting the involvement of focal adhesion (FA)-associated exocytosis in the release of WPBs. For the first time, we demonstrate a regulatory role for the FA-resident guanine nucleotide exchange factor (GEF) {beta}-PIX in VWF secretion. Through its GEF activity toward Cdc42 and Rac, {beta}-PIX activates p21-activated kinases, driving the necessary cytoskeletal dynamics. Disruption of {beta}-PIX function results in abnormal cytoskeletal architecture that fails to respond appropriately to phorbol ester stimulation. Consequently, F-actin, nonmuscle myosin IIA (NMIIA), and septins become mislocalised, forming prominent filaments above the nucleus. These data provide a novel insight into the spatial targeting and molecular events underpinning the exocytosis of endothelial secretory organelles. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/652370v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1dbc615org.highwire.dtl.DTLVardef@1a9a959org.highwire.dtl.DTLVardef@1ad31aforg.highwire.dtl.DTLVardef@1990000_HPS_FORMAT_FIGEXP M_FIG C_FIG