A new green fluorescent protein construct for localizing and quantifying peptide release.

Abstract

Green fluorescent protein (GFP)-tagged secretory proteins recently have been used for studying packaging of peptides, secretory vesicle dynamics, and regulation of peptide release. In cells in which release occurs from sites with an abundance of vesicles, it is difficult to resolve the exact location of individual exocytotic events with standard wide-field epifluorescence microscopy. Furthermore, current GFP constructs are not well suited for real-time measurement of peptide release from large numbers of cells. Here, we describe a new pH-sensitive construct that is designed for localizing and quantifying release of neuropeptides and peptide hormones. Specifically, the yellow GFP variant called Topaz was fused to proAtrial natriuretic peptide (proANP, also called proAtrial natriuretic factor). The fluorescence of this fusion protein is low in normally acidic secretory vesicles but increases approximately 10-fold upon neutralization. Furthermore, it is released upon depolarization of PC12 cells. Finally, individual release events can be detected as brief localized flashes of fluorescence. ProANF-Tpz should prove useful for studying single release events by wide-field epifluorescence microscopy and for fluorometer-based real-time peptide release measurements from high numbers of cells.