Content delivery to newly forming Weibel-Palade bodies is facilitated by multiple connections with the Golgi apparatus

Abstract

Key Points• WPBs stay connected to the Golgi apparatus until vesicle formation is completed.• During biogenesis at the Golgi, WPBs increase in size through the addition of nontubular VWF.Weibel-Palade bodies (WPBs) comprise an on-demand storage organelle within vascular endothelial cells. It's major component, the hemostatic protein von Willebrand factor (VWF), is known to assemble into long helical tubules and is hypothesized to drive WPB biogenesis. However, electron micrographs of WPBs at the Golgi apparatus show that these forming WPBs contain very little tubular VWF compared with mature peripheral WPBs, which raises questions on the mechanisms that increase the VWF content and facilitate vesicle growth. Using correlative light and electron microscopy and electron tomography, we investigated WPB biogenesis in time. We reveal that forming WPBs maintain multiple connections to the Golgi apparatus throughout their biogenesis. Also by volume scanning electron microscopy, we confirmed the presence of these connections linking WPBs and the Golgi apparatus. From electron tomograms, we provided evidence that nontubular VWF is added to WPBs, which suggested that tubule formation occurs in the WPB lumen. During this process, the Golgi membrane and clathrin seem to provide a scaffold to align forming VWF tubules. Overall, our data show that multiple connections with the Golgi facilitate content delivery and indicate that the Golgi appears to provide a framework to determine the overall size and dimensions of newly forming WPBs. (Blood. 2015;125(22):3509-3516) IntroductionRapid secretion of the endothelial storage organelles, the WeibelPalade bodies (WPBs), 1 is fundamental for hemostasis. WPBs contain the hemostatic glycoprotein von Willebrand factor (VWF), which recruits platelets to sites of injury to arrest bleeding.2 Within WPBs, VWF is packed into helical tubules that give the organelle an elongated shape with a length of 1 to 5 mm and a width of 100 to 300 nm. 1,[3][4][5] The formation of WPBs is dependent on VWF and also occurs on VWF expression in nonendothelial cells. 6,7 VWF is synthesized in the endoplasmic reticulum as a pre-proprotein consisting of a signal peptide, a propeptide, and mature VWF.2 On removal of the signal peptide, VWF dimers are formed that are transported to the Golgi apparatus. At the Golgi, the propeptide is cleaved from mature VWF to guide multimerization and tubule formation. 3,8,9 VWF tubule formation is crucial for the development of mature, densely packed elongated WPBs. Mutations in the VWF gene, as found in patients with the bleeding disorder von Willebrand disease, were shown to result in altered WPB morphology. 2,[10][11][12] In vitro studies on VWF tubule formation demonstrated that the core of the VWF tubules is formed by the propeptide and the N-terminal D9 and D3 assembly of mature VWF.3 However, it is still poorly understood how the formation of the VWF tubules is related to WPB biogenesis.Electron microscopy studies have revealed several stages in the WPB formation pro...

von Willebrand factor is dimerized by protein disulfide isomerase

“…15 Then we observed the recovery of WPB formation in the absence and presence of the PDI inhibitors 16F16 21 and PAO. 22 As described previously, 23 the first small WPB-like structures were observed after 4 h ( Figure 4C), and full recovery of cigar-shaped WPBs was reached after 8 h in the absence of PDI inhibitors ( Figure 4F). In contrast, WPB formation was completely abolished in cells treated with 16F16 ( Figure 4D,G) and strongly reduced in the PAO-treated cells ( Figure 4E,H), indicating that VWF dimerization was inhibited, leading to loss of VWF multimers, and thus WPBs.…”

Fifth Åland Island conference on von Willebrand disease

“…Visualizing the life cycle of WPB s at the ultrastructural level . A, The life cycle of WPB s. B, Correlative light and electron microscopy of nascent WPB formation.…”

“…To elucidate the mechanistic details of the WPB life cycle, biogenesis of WPBs was followed using various ultrastructure imaging techniques (Figure B‐D). WPBs were first depleted from human umbilical cord venous endothelial cells (HUVECs) by phorbol 12‐myristate 13‐acetate (PMA) treatment, which induces exocytosis of WPBs . Synthesis of nascent WPBs over time was then followed by visualization of VWF.…”

Quantifying Golgi structure using EM: combining volume-SEM and stereology for higher throughput

“…However, LM lacks the resolution needed for visualising the structural elements of the Golgi directly (Durisic et al 2014). EM is a more suitable tool because it can visualise membranes, tubules, vesicles, (Amos and Grimstone 1968; Lucocq et al 1995; Trucco et al 2004; Martínez-Alonso et al 2013; Beznoussenko et al 2014; Mourik et al 2015) as well as fine details such as structural stages in vesicle and cisternal biogenesis (Amos and Grimstone 1968; Lucocq et al 1995; Pellet et al 2013; Martínez-Alonso et al 2013). EM also displays the Golgi in a cellular context allowing simultaneous study of related membrane traffic organelles such as ER and plasma membrane.…”