Weibel‐Palade bodies (WPBs) are endothelial‐specific, rod‐shape secretory organelles that, upon stimulation, exocytose to release strings of the adhesive glycoprotein von Willebrand Factor (VWF). These strings are required to initiate haemostasis by recruiting platelets to sites of vascular injury. Additionally, WPB exocytosis results in translocation of the transmembrane protein P‐selectin to the cell surface, promoting leukocyte recruitment to inflamed tissue. WPB formation and regulated secretion is therefore central to endothelial function. This thesis describes the development of a high‐content RNAi screen aimed at identifying novel regulators of these processes, as well as the subsequent validation and characterisation of one candidate gene, G protein‐coupled receptor (GPCR) kinase 2 (GRK2). In human umbilical vein endothelial cells (HUVECs), GRK2‐depletion caused a 30% reduction in WPB numbers by increasing steady‐state release of VWF. GRK2 attenuates GPCR‐mediated signalling through phosphorylation of agonist‐occupied receptors. It was therefore hypothesised that this phenotype may reflect an increased sensitivity to stimulus. Indeed, activation of GRK2‐depleted HUVECs with the GPCR agonist histamine resulted in a two‐ and three‐fold increase in VWF secretion and P‐selectin dependent recruitment of THP‐1 cells respectively. Calcium signalling was also enhanced, consistent with impaired receptor desensitisation. These studies have therefore uncovered a novel anti‐inflammatory role for endothelial GRK2. WPB biogenesis at the trans‐Golgi network depends on recruitment of an AP‐1/clathrin coat to support the tubulation of VWF multimers. Preliminary work presented here suggests loss of clathrin heavy chain or light chain B results in the formation of shorter organelles, whilst light chain A depletion promotes the biogenesis of longer granules. Clathrin components may therefore differentially regulate WPB formation. Finally, a preliminary model for WPB membrane retrieval following exocytosis (necessary for the maintenance of cell size homeostasis) is presented. This entails closure of the fusion pore and the subsequent recycling of the internalised membrane by clathrin‐mediated vesiculation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:626769 |
| Date | January 2014 |
| Creators | Stevenson, N. L. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1418704/ |
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