Real-time imaging of the dynamics and secretory behavior of Weibel-Palade bodies.
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).


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.
Romani de Wit
Thalia
T
Department of Plasma Proteins, Sanquin Research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
Rondaij
Mariska G
MG
Hordijk
Peter L
PL
Voorberg
Jan
J
van Mourik
Jan A
JA
D002118:Calcium / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003199:Computer Systems; D000242:Cyclic AMP / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects; D022001:Focal Adhesions; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D020302:Weibel-Palade Bodies / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D049452:Green Fluorescent Proteins; D005576:Colforsin; D000242:Cyclic AMP; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium