Endocytosis and Exocytosis Events Regulate Vesicle Traffic in Endothelial Cells |
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Authors: | WD Niles AB Malik |
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Institution: | (1) Department of Pharmacology, University of Illinois College of Medicine, 835 South Wolcott Avenue (M/C 868), Chicago, IL 60612, USA, US |
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Abstract: | We used water-soluble styryl pyridinium dyes that fluoresce at the membrane-water interface to study vesicle traffic in endothelial
cells. Cultured endothelial cells derived from bovine and human pulmonary microvessels were incubated in styryl probes, washed
to remove dye from the plasmalemmal outer face, and observed by digital fluorescence microscopy. Vesicles that derived from
plasmalemma by endocytosis were filled with the styryl dye. These vesicles were distributed throughout the cytosol as numerous
particles of heterogeneous diameter and brightness. Vesicle formation was activated 2-fold following addition of extracellular
albumin whereas a control protein, immunoglobulin G, had no effect. Dye uptake was abrogated by labeling at low temperatures
and inhibitors of phosphoinositide-3-kinase (PI 3-kinase). Tyrosine kinase inhibitors (genistein and herbimycin A) prevented
the albumin-induced vesicle formation. Cytochalasin B prevented vesicle redistribution indicating involvement of actin filaments
in translocation of endosomes away from sites of vesicle formation. Styryl dye was lost from cells by exocytosis as evident
by the disappearance of discrete fluorescent particles. N-ethylmaleimide and botulinum toxin types A and B caused cells to accumulate increased number of vesicles suggesting that exocytosis was regulated by NSF-dependent
SNARE mechanism. The results suggest that phosphoinositide metabolism regulates endocytosis in endothelial cells and that
extracellular albumin activates endocytosis by a mechanism involving tyrosine phosphorylation, whereas exocytosis is a distinct
process regulated by the SNARE machinery. The results support the hypothesis that albumin regulates its internalization and
release in vascular endothelial cells via activation of specific endocytic and exocytic pathways. |
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Keywords: | : Styryl pyridinium dyes — Digital fluorescence microscopy — Vesicle dynamics |
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