Ca2+-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca2+-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca2+ level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca2+-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles. (C) 2007 Elsevier Inc. All rights reserved.