Streetley, JWA; (2014) Cryo-electron microscopy of Weibel-Palade Bodies in human endothelial cells. Doctoral thesis , UCL (University College London).

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Abstract

Weibel-Palade bodies (WPBs) are a secretory storage organelle found in endothelial cells. Their primary cargo is the large haemostatic protein von Willebrand factor (VWF), however there are many other minor components, both in the membrane and lumen of the WPB. VWF is stored in long helical tubules which define the ‘rod-like’ shape of the WPBs. WPBs are released in response to raised intracellular Ca²⁺ or cAMP levels, secreting the VWF content. Upon exocytosis, the environment of the VWF changes from acidic to neutral pH, causing the tubules of VWF to unfurl into strings. The expansion of the VWF tubules into strings exposes binding and cleavage sites, turning VWF into its haemostatically active form. As tubules transform into strings, the overall shape of the WPB changes, first becoming round before releasing its contents. Imaging at the periphery of frozen-hydrated, whole human umbilical vein endothelial cells (HUVECs) by cryo-electron microscopy (cryo-EM) and cryo-electron tomography has been used to study WPBs within their native environment. The structure of WPBs within HUVECs both unstimulated and stimulated to release WPBs with raised intracellular Ca²⁺ has been examined. This has enabled imaging of both rod-shaped and rounded WPBs. Round WPBs in HUVECs exposed to hypotonic shock and within human heart microvasculature endothelial cells (HHMECs) have also been imaged. A subset of the WPBs imaged within endothelial cells contain an internal vesicle within them. Light microscopy studies have shown a vesicle with corresponding characteristics is enriched for the membrane protein CD63, a known constituent of WPBs. Finally, a technique for chemically fixing cells for light microscopy studies prior to plunge-freezing is shown, with subsequent correlative light and electron microscopy experiments looking at CD63-enriched vesicles within WPBs. The quality of preservation for cryo-EM experiments, including tomography and image processing for correlative microscopy is discussed.

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