2117 Research Article Introduction Weibel-Palade bodies (WPBs), first described 40 years ago (Weibel and Palade, 1964), are among the most striking objects to be seen in cells. As long cigar-shaped granules, these lysosome-related organelles of endothelial cells play a central role in the initiation of inflammation and in haemostasis. Apart from their overall shape, their hallmark has been the presence of internal striations running parallel to their long axis that are revealed as tubules in transverse section (Weibel and Palade, 1964). These tubules are von Willebrand Factor (VWF), a key haemostatic mediator, which is stored within the organelle (Wagner et al., 1982). We have demonstrated that the storage of VWF as tubules is important for this protein’s ability to trap platelets (Michaux et al., 2006). Only when the tubules are intact can the recently described long strings of platelet- catching VWF (Dong et al., 2002) unfurl efficiently from the endothelial cells upon exocytosis. Deliberate disruption of the VWF tubules not only reduces platelet recruitment, but also causes rounding of the organelles (Michaux et al., 2006). We have therefore defined the VWF tubule as key to both WPB ultrastructure and VWF function. Despite their importance in driving the formation of WPB shape, the relationship between tubule formation and WPB biogenesis is poorly understood. A great deal is known about the complex biosynthesis of VWF, including its cleavage into a pro-peptide plus mature protein by furin, and the need for interactions between these two components for tubule formation to occur (reviewed in Hannah et al., 2002; Michaux and Cutler, 2004; van Mourik et al., 2002); however, even simple questions, such as exactly where along the secretory pathway the tubules start to drive the formation of this uniquely-shaped organelle, remain unanswered. A second area of interest concerns the role of clathrin coats in WPB formation. Although we have recently proposed a novel scaffolding role for an AP-1/clathrin coat in initial WPB formation (Lui-Roberts et al., 2005), the relationship between the AP-1/clathrin coat and the formation of tubules themselves has yet to be investigated. This has become crucial since we identified tubule formation as the key to WPB formation. In addition, clathrin-coated vesicles are used for removing missorted material from the post-trans-Golgi network (TGN) but are still immature in endocrine and neuroendocrine cells (Dittie et al., 1999; Klumperman et al., 1998). It is thought that control of entry into forming granules at the TGN (‘sorting by entry’) is not completely effective at excluding proteins destined for other post-TGN destinations, such as the endosomal system or constitutive exocytosis. The post-budding sorting step (‘sorting by retention’) allows the removal of such missorted proteins (reviewed in Arvan and Castle, 1998; Borgonovo et al., 2006). There is no evidence that clathrin- 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. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/12/2117/DC1 Key words: High-pressure freezing, Weibel-Palade bodies, von Willebrand Factor Summary High-pressure freezing provides insights into Weibel-Palade body biogenesis Helen L. Zenner 1, *, Lucy M. Collinson 2, * ,‡ , Grégoire Michaux 1,§ and Daniel F. Cutler 1,¶ 1 MRC Laboratory of Molecular Cell Biology, Cell Biology Unit, and Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK 2 Electron Microscopy Facility, MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, UK *These authors contributed equally to this work ‡ Present address: Electron Microscopy Unit, Cancer Research UK London Research Institute Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK § Present address: UMR 6061, Faculté de Médecine, 2 avenue du Professeur Léon Bernard, CS 34317, 35043 Rennes Cedex, France ¶ Author for correspondence (e-mail: d.cutler@ucl.ac.uk) Accepted 20 April 2007 Journal of Cell Science 120, 2117-2125 Published by The Company of Biologists 2007 doi:10.1242/jcs.007781 Journal of Cell Science JCS ePress online publication date 29 May 2007