TY - JOUR
T1 - Real-time imaging of the dynamics and secretory behavior of Weibel-Palade bodies
AU - De Wit, Thalia Romani
AU - Rondaij, Mariska G.
AU - Hordijk, Peter L.
AU - Voorberg, Jan
AU - Van Mourik, Jan A.
PY - 2003/5/1
Y1 - 2003/5/1
N2 - 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.
AB - 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.
KW - Endothelial cells
KW - Green fluorescent protein
KW - Real-time imaging
KW - Von Willebrand factor
KW - Weibel-Palade bodies
UR - http://www.scopus.com/inward/record.url?scp=0038705032&partnerID=8YFLogxK
U2 - https://doi.org/10.1161/01.ATV.0000069847.72001.E8
DO - https://doi.org/10.1161/01.ATV.0000069847.72001.E8
M3 - Article
C2 - 12676800
SN - 1079-5642
VL - 23
SP - 755
EP - 761
JO - Arteriosclerosis, Thrombosis, and Vascular Biology
JF - Arteriosclerosis, Thrombosis, and Vascular Biology
IS - 5
ER -