on April 3, 2003
Published online before print April 3, 2003, doi: 10.1161/01.ATV.0000069847.72001.E8
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Submitted on January 19, 2003
Accepted on March 12, 2003
Real-Time Imaging of the Dynamics and Secretory Behavior of Weibel-Palade Bodies
Thalia Romani de Wit ;
From the Departments of Plasma Proteins (T.R.d.W., M.G.R., J.V., J.A.v.M.) and Experimental Immunohaematology (P.L.H.), Sanquin Research at CLB, Amsterdam, and the Department of Vascular Medicine (J.A.v.M.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
* To whom correspondence should be addressed. E-mail: J_van_Mourik{at}clb.nl.
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 [Ca2+]i- or cAMP-raising secretagogues, membrane-apposed patches were formed, suggesting fusion of WPBs with the plasma membrane. Patches remained visible for >20 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.
Conclusions--In conclusion, the fusion protein vWF-GFP provides a powerful tool to study, in real time, signal-mediated trafficking of WPBs.
Key words: von Willebrand factor • Weibel-Palade bodies • endothelial cells • real-time imaging • green fluorescent protein
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