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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.

SUBMITTER: Berriman JA

PROVIDER: S-EPMC2765093 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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

Berriman John A JA Li Sam S Hewlett Lindsay J LJ Wasilewski Sebastian S Kiskin Fedir N FN Carter Tom T Hannah Matthew J MJ Rosenthal Peter B PB

Proceedings of the National Academy of Sciences of the United States of America 20090929 41

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 r ...[more]

PMID: 19805028

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Project description:Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation. The molecules that contribute to these processes are co-stored in secretory granules. How can the cells control release of granule content to allow differentiated responses? Selected agonists recruit an exocytosis-linked actin ring to boost release of a subset of cargo.SummaryBackground Endothelial cells harbor specialized storage organelles, Weibel-Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P-selectin. During full fusion, secretion of this large hemostatic protein and smaller pro-inflammatory proteins are thought to be inextricably linked. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high-throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins. Results Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms. Conclusions Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo-content release and the treatment of patients with von Willebrand disease.

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

Publications

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

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