High-pressure freezing provides insights into Weibel-Palade body biogenesis.

The Weibel-Palade bodies (WPBs) of endothelial cells play an important role in haemostasis and the initiation of inflammation, yet their biogenesis is poorly understood. Tubulation of their major content protein, von Willebrand factor (VWF), is crucial to WPB function, and so we investigated further the relationship between VWF tubule formation and WPB formation in human umbilical vein endothelial cells (HUVECs). By using high-pressure freezing and freeze substitution before electron microscopy, we visualised VWF tubules in the trans-Golgi network (TGN), as well as VWF subunits in vesicular structures. Tubules were also seen in WPBs that were connected to the TGN by membranous stalks. Tubules are disorganised in the immature WPBs but during maturation we found a dramatic increase in the spatial organisation of the tubules and in organelle electron density. We also found coated budding profiles suggestive of the removal of missorted material after initial formation of these granules. Finally, we discovered that these large, seemingly rigid, organelles flex at hinge points and that the VWF tubules are interrupted at these hinges, facilitating organelle movement around the cell. The use of high-pressure freezing was vital in this study and it suggests that this technique might prove essential to any detailed characterisation of organelle biogenesis.