Real-Time Imaging of the Dynamics and Secretory Behavior of Weibel-Palade Bodies

Abstract

Objective-Weibel-Palade bodies (WPBs) are specialized secretory granules found in endothelial cells. These vesicles store hormones, enzymes, and receptors and exhibit regulated exocytosis on cellular stimulation. Here we have directly visualized intracellular trafficking and the secretory behavior of WPBs in living cells by using a hybrid protein consisting of von Willebrand factor (vWF), a prominent WPB constituent, and green fluorescent protein (GFP). Methods and Results-Immunofluorescence microscopy demonstrated that this chimera was targeted into WPBs. In resting cells, some WPBs seemed motionless, whereas others moved at low speed in a stochastic manner. On stimulation of cells with [Ca 2ϩ ] i -or cAMP-raising secretagogues, membrane-apposed patches were formed, suggesting fusion of WPBs with the plasma membrane. Patches remained visible for Ͼ20 minutes. This sustained, membrane-associated retention of vWF might play a role in focal adhesion of blood constituents to the endothelium after vascular injury. In addition, stimulation with cAMP-raising agonists resulted in clustering of a subset of WPBs in the perinuclear region of the cell. Apparently, these WPBs escaped secretion. This feature might provide a mechanism to control regulated exocytosis. V ascular endothelial cells are equipped with a machinery that, on perturbation, allows prompt delivery of a number of bioactive substances, including hormones, receptors, and adhesive molecules, to the surface of the cell. A distinct subset of proteins destined to be released on stimulation of the endothelium stems from Weibel-Palade bodies (WPBs), typical and morphologically highly organized storage vesicles that release their contents by regulated exocytosis. WPBs are endothelial cell-specific elongated organelles, enclosed by a limiting membrane, which are Ϸ0.1 m wide and up to 4 m long 1 and originate from the trans-Golgi network. 2,3 They serve as storage vesicles for a variety of proteins with different biologic functions, such as the leukocyte adhesion receptor P-selectin 4,5 and the chemokine interleukin-8 (IL-8). 6,7 Effective translocation of P-selectin from WPBs to the cell surface is critical for the binding and rolling of leukocytes on the endothelium at sites of inflammation. 8,9 Similarly, regulated exocytosis of IL-8 provides an effective means for controlling local leukocyte extravasation. 10 One of the most prominent WPB residents is von Willebrand factor (vWF), an adhesive multimeric glycoprotein that contributes to platelet adhesion and hemostatic plug formation at sites of vascular injury (reviewed in Sadler 11 and Ruggeri 12 ). Regulated secretion of vWF provides an adequate means for endothelial cells to actively participate in controlling the arrest of bleeding after vascular damage. Thus, it seems likely that regulated exocytosis of WPBs serves several physiological functions, including inflammatory and hemostatic responses. Conclusions-In See coverRegulated exocytosis of vWF and other WPB residents involves the translocation of WPBs ...

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