Chapter 4 - Correlative Light Microscopy and Electron Tomography to Study Von Willebrand Factor Exocytosis from Vascular Endothelial Cells

Abstract

Revealing the ultrastructure and function of fluorescently labeled cellular components by correlative light and electron microscopy (CLEM) facilitates the study of structure-function relationships in complex biological processes. Given the diversity of available fluorescent tags, light microscopy is ideal for monitoring dynamic cellular processes, while electron microscopy reveals the morphological context of structures at high resolution. Endothelial cells lining the blood vessel wall contain storage organelles called Weibel-Palade bodies (WPBs), which contain tubules of densely packed helical spirals of the blood coagulation protein Von Willebrand factor (VWF). Exocytosis of WPBs is triggered upon vascular damage and results in the transformation of stored tubular VWF into secreted extracellular VWF. Upon exocytosis, VWF rearranges into long filamentous strings to recruit platelets from the blood. During this secretion process, large intracellular VWF exocytosis structures are formed called secretory pods. Here, we describe a CLEM method used to study the relationship between the secretory pod and secreted VWF where confocal microscopy on whole cells was combined with serial electron tomography on chemically fixed, plastic-embedded sections. We show that the combination of these two well-established microscopy modalities provides a robust and generic CLEM method suitable for the characterization of VWF secretion sites.