The Weibel Palade body (WPB) is the major regulated secretory organelle of endothelial cells. The WPB is used here as a model system to investigate the formation and acquisition of membrane identity of regulated secretory organelles. Heterologous expression of von Willebrand (VWF), the main secretory cargo of WPBs, in cells lacking endogenous VWF, is able to induce the formation of structures that are not only morphologically indistinguishable from WPBs but also recruit the appropriate membrane components resulting in the correct membrane identity. The question arises as to how VWF acts within the lumen of the secretory pathway to do this. The working hypothesis is that VWF drives the formation of WPB and the acquisition of membrane identity by direct interaction with specific lipids on the lumenal face of the lipid bilayer. This thesis describes attempts to test this hypothesis both directly, by looking for putative interactions of VWF with membrane lipids and indirectly, by studying the role of glycosphingolipids in WPB formation. To look for direct interaction of VWF with membrane lipids, attempts were made to produce recombinant, enzymatically tagged VWF probes and develop an overlay assay. Glycoarrays were also probed with VWF to identify potential glycan binding partners. VWF was found to bind to a class of fucosylated glycans on the array. In order to look for the influence of glycosphingolipids on WPB formation two distinct approaches were taken The effect of drug-mediated inhibition of membrane glycosphingolipid synthesis on WPB formation in cultured human endothelial cells was investigated. Inhibition of glucosylceramide synthesis, and hence complex glycosphingolipids, by N-butyldeoxygalactonojirimycin had no apparent effect on VWF trafficking. Trafficking of heterologously expressed VWF was studied in two epithelial cell lines, known to have distinct glycosphingolipid compositions. The two cell lines were shown to differ in VWF trafficking both morphologically and biochemically. VWF was also shown to be differentially glycosylated between the two cell lines, suggesting a link between VWF glycosylation and trafficking.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:505287 |
| Date | January 2009 |
| Creators | Houston, Karen Grace |
| 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/17253/ |
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