Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells

Abstract

In endothelial cells, the multifunctional blood glycoprotein von Willebrand Factor (VWF) is stored for rapid exocytic release in specialized secretory granules called Weibel-Palade bodies (WPBs). Electron cryomicroscopy at the thin periphery of whole, vitrified human umbilical vein endothelial cells (HUVECs) is used to directly image WPBs and their interaction with a 3D network of closely apposed membranous organelles, membrane tubules, and filaments. Fourier analysis of images and tomographic reconstruction show that VWF is packaged as a helix in WPBs. The helical signature of VWF tubules is used to identify VWF-containing organelles and characterize their paracrystalline order in low dose images. We build a 3D model of a WPB in which individual VWF helices can bend, but in which the paracrystalline packing of VWF tubules, closely wrapped by the WPB membrane, is associated with the rod-like morphology of the granules.electron cryomicroscopy ͉ paracrystal ͉ von Willebrand factor ͉ tomography E ndothelial cells line the inner surfaces of blood vessels and play important roles in hemostasis, thrombosis, and inflammation. Some of these roles are achieved by secretion of the large, multimeric blood glycoprotein von Willebrand factor (VWF). VWF has multiple ligands and on acute release functions as an adhesive protein to bind platelets to sites of vascular injury. VWF circulating in the bloodstream also functions as a carrier for coagulation Factor VIII, increasing its lifetime. Defects in VWF and its storage are responsible for bleeding disorders including von Willebrand's disease (1).VWF is synthesized as a 350-kDa precursor (proVWF) that forms disulfide-linked dimers in the ER through its C-terminal cysteine knot domain. Proteolytic cleavage of proVWF in the Golgi gives rise to the N-terminal propolypeptide (a 100-kDa protein called proregion) and to mature VWF dimers that form large homo-oligomers through disulfide-links near each of its mature N-termini, a process catalyzed by proregion (2, 3). VWF and proregion remain non-covalently associated and are stored together in specialized secretory organelles called WeibelPalade bodies (WPBs), first identified by EM of fixed tissue sections as rod-shaped organelles containing fine tubules (4). Secretagogues stimulate WPB exocytosis, releasing VWF and other low molecular weight molecules such as cytokines and chemokines into the bloodstream (5), although mature VWF and its proregion account for greater than 95% of the protein in the granule (6). On release, VWF multimers are able to unfurl to strings up to 100 m long and associate with multiple ligands on platelet and endothelial cell surfaces at the site of vascular injury to help form a platelet plug. Mechanical shear exposes ligand binding sites on VWF as well as sites for cleavage by the protease ADAMTS13, which regulates the length of VWF multimers in the bloodstream (7).Like most other secretory granules, WPBs are thought to form at the trans-Golgi network (TGN) in a pH-dependent process. P-selectin is also recru...

Protein mobilities and P-selectin storage in Weibel–Palade bodies

“…Tight packing of tubules in the paracrystal could introduce a 'molecular sieve' effect (Papadopoulos et al, 2000) introducing a size dependence for protein diffusion. The largest density gaps in the WPB lumen are the interiors of the tubules themselves (inner diameter of ~12 nm), and the 4-8 nm spaces between the tubules (Berriman et al, 2009). Lateral fenestrations of ~7 nm in the tubule walls may connect tubule interiors (Berriman et al, 2009), providing a route for percolative diffusion of small molecules.…”

“…The largest density gaps in the WPB lumen are the interiors of the tubules themselves (inner diameter of ~12 nm), and the 4-8 nm spaces between the tubules (Berriman et al, 2009). Lateral fenestrations of ~7 nm in the tubule walls may connect tubule interiors (Berriman et al, 2009), providing a route for percolative diffusion of small molecules. Near the percolation threshold, diffusion decreases steeply with the decrease in free WPB volume and increased size of diffusing molecules (Novak et al, 2009), so small decreases in free volume may cause dramatic mobility changes.…”

“…Electron tomography of mature WPBs in vitrified HUVECs shows that the lumen comprises a paracrystalline packing of Proregion-VWF tubules (Berriman et al, 2009). Paracrystal formation is linked to acidification and dehydration of the WPBs, and possible direct interactions between tubules and the membrane.…”

“…This includes budding of clathrin-coated vesicles from immature WPBs (Zenner et al, 2007) providing a mechanism for removal of missorted proteins and components not destined for regulated secretion (Arvan and Castle, 1998;Katsumata et al, 2007;Klumperman et al, 1998) and organelle acidification (Erent et al, 2007). Dehydration of organelle content is indicated by a high buoyant density of mature WPBs (Ewenstein et al, 1987) and a membrane tightly apposed to the paracrystalline core (Berriman et al, 2009). WPBs therefore undergo processes common to RSO biogenesis in other cells but uniquely, their size and morphology provide an opportunity to quantitatively analyse protein mobilities in individual RSOs in live cells.…”

“…Endothelial cells contain large rod-like secretory granules, the Weibel-Palade bodies (WPBs) (Weibel and Palade, 1964). WPB formation is driven by von Willebrand factor (VWF), and their shape is the result of a paracrystalline matrix of tubules composed of the cleaved VWF propolypeptide (Proregion) and mature VWF (Berriman et al, 2009). WPB formation at the trans-Golgi network (TGN) requires a membrane scaffold of AP-1 and clathrin (Lui-Roberts et al, 2005), although in other respects WPB maturation proceeds similarly to other RSOs (Tooze et al, 2001).…”