A novel tool to visualize the cell secretory pathway. Focus on 'A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK_1 cells expressing aquaporin-2'

摘要

a number of fluorescence-based techniques have been devel-oped and used to visualize the trafficking and exocytosis of vesicles in living cells and intact tissues. Several of these techniques are based on the addition of exogenous fluorescent compounds that label the secretory vesicles through endocyto-sis or by acidotropic dye trapping. For example, FM dyes (Molecular Probes/Invitrogen) have been used for the past 15 years to study the dynamics of vesicle trafficking in a variety of cell types (1). These styryl dyes are essentially nonfluores-cent when in solution but brighten markedly when inserted in membranes. After vesicle preloading by stimulated exocytosis-endocytosis, the FM dyes are released subsequent to a stimulus by exocytosis, which can be readily documented as a decrease in fluorescence. In acidotropic dye trapping, the acidotropic fluorophores, such as quinacrine, quickly penetrate cell mem-branes because a fraction of the molecules is uncharged at physiological pH, and then accumulate in acidic cell organelles where they are "trapped" after becoming protonated and are no longer membrane permeable. The dyes are rapidly released on exocytosis, as has been used to document secretion of ATP secretory vesicles (2) and renin granules in the kidney (5). Unfortunately, most of these dyes are not specific for secretory vesicles as they also label the lysosomes of the degradation pathway and even cell nuclei (5), nor do they directly label the secreted, cell-specific vesicle cargo itself, like neurotransmit-ters, insulin, and renin.