Imaging von Willebrand Factor secretion in vascular endothelial cells using live-cell imaging and correlative light and electron microscopy

Vascular endothelial cells lining the inner surface of blood vessels synthesize high molecular weight multimers of the blood coagulation protein von Willebrand factor (VWF). At the trans Golgi network, VWF multimers are folded together with the VWF propeptide (also called proregion) into tubular structures for storage in cigar-shaped organelles. These organelles, called Weibel-Palade bodies (WPBs), are 1-3μm long and 0.1-0.3μm wide and mature in the periphery of the cell by recruiting proteins to make them responsive to stimulation [1,2]. Stimulation occurs due to vascular injury, shear stress or hypoxia and results in the exocytosis of WPBs releasing their content into the bloodstream. VWF is then transformed into long VWF strings which capture platelets and induce primary hemostasis. The typical tubular packaging of VWF was often suggested to be required for successful secretion as agents disrupting the tubular organization of WPBs also affected VWF string formation upon stimulation [3]. It was therefore hypothesized that VWF is stored as a compressed spring within the WPBs. Upon fusion the VWF would then pop out of the cells due to a rapid switch in pH, resulting in the direct unfolding of the protein into VWF strings [3,4]. Recently we described a novel VWF containing structure in cultured vascular endothelial cells which is formed upon stimulation [5]. We refer to this structure as a “secretory pod” since it seems to derive from multiple WPBs and was identified as a VWF release site. With transmission electron microscopy (TEM) we showed that this structure is a membrane-delimited structure containing filamentous material resembling unfurled VWF. This suggests that the tubular packaging of VWF in WPBs is not required for secretion and string formation. To study the origin of secretory pods and the remodeling of VWF into strings, we applied several imaging techniques. We visualized secretory pod formation by performing live-cell imaging on stimulated endothelial cells expressing proregion-GFP to label the WPBs. As WPB secretion was found to be very rapid we used very short image intervals. We were able to detect that the coalescence of multiple WPBs indeed resulted in the formation of a secretory pod as shown in figure 1. We employed Correlative Light and Electron Microscopy (CLEM) to study VWF remodeling into strings upon secretion from secretory pods. For this technique we combined Confocal Scanner Laser Microscopy (CLSM) with conventional TEM. We correlated confocal pictures, showing pods and strings, to consecutive EM sections. VWF released from secretory pods appears as a globular mass before it is remodeled into VWF strings (figure 2). In addition, 3D reconstructions obtained by electron tomography revealed a connection of secreted VWF with the secretory pod membrane. This connection suggests the presence of an anchoring site which may help the protein unfurl. In contrast to previous hypotheses that tubular packaging of VWF is required for string formation, we show that WPBs can coalesce to form secretory pods upon stimulation. Moreover, VWF released from secretory pods forms a globular mass of protein showing emerging VWF strings and suggesting that remodeling of VWF into strings might occur independently from secretion in a process initiated by fluid flow [6].