Characterisation of the Weibel-Palade body fusion pore using optical and electrochemical techniques

Endothelial cells (ECs) form a dynamic surface within blood vessels, constantly monitoring and responding to their local environment by synthesising and releasing diffusible and cell surface bioactive molecules. The regulated secretion of a range of such molecules is mediated by the exocytosis of secretory granules (SGs) called Weibel-Palade bodies (WPBs). Optical data has suggested that WPBs undergo different forms of exocytosis whereby subsets of cargo molecules are released or retained based on their size. In other cell types the SG fusion pore is implicated in regulating selective release of specific molecules, however almost nothing is known about the WPB fusion pore. A highly effective method for studying fusion pore formation and expansion is carbon fibre amperometry, an electrochemical technique, allowing each stage of exocytosis to be quantified with submillisecond resolution. The primary aim of this thesis was therefore to establish carbon fibre amperometry as a technique to measure the WPB fusion pore in order to analyse its dynamics for the first time. Because WPBs did not contain endogenous oxidisable molecules suitable for amperometric detection, methods were developed to specifically load WPBs with suitable molecules. Amperometry was then used in combination with live cell imaging of WPB exocytosis to provide direct characterisation of the properties of the WPB fusion pore during Ca2+-driven WPB exocytosis. WPB fusion pore parameters were comparable to those reported for SGs in other cell types (e.g. chromaffin cells), indicating that WPBs may share similar processes controlling membrane fusion and the mobilisation and release of oxidisable species. Half of the exocytotic events demonstrated a pre-spike foot (PSF) signal prior to the current spike indicating that a restricted fusion pore had initially formed. In rare cases PSF signals showed step changes or fluctuations suggesting that during expansion the fusion pore may transition through different configurations before fully opening. Following characterisation of the WPB fusion pore under control conditions, factors which may affect the behaviour of the fusion pore were investigated, including the role of PM cholesterol. In line with previous studies, depletion of PM cholesterol increased the rate of fusion pore expansion and decreased the duration of the lifetime of the restricted fusion pore. This has been attributed to the promotion of the formation of a restricted fusion pore due to the intrinsic negative curvature of cholesterol, which is subsequently destabilised upon removal of cholesterol. Results presented here therefore support this idea. However, in contrast to results obtained from a range of cell types, which reported an inhibition of SG exocytosis following PM cholesterol depletion, WPB exocytosis remained largely unperturbed providing evidence for the insensitivity of the EC response to removal of PM cholesterol. In conclusion, the work presented in this thesis provides a detailed characterisation of the WPB fusion pore and has begun to address factors which may be important for its regulation. The extensive characterisation under control conditions now allows further elements potentially involved in the regulation of the WPB fusion pore to be investigated.