TARGETING OF GREEN FLUORESCENT PROTEIN TO NEUROENDOCRINE SECRETORY GRANULES - A NEW TOOL FOR REAL-TIME STUDIES OF REGULATED PROTEIN SECRETION

Human chromogranin B (hCgB), a soluble marker protein of neuroendocrin e secretory granules, was fused to green fluorescent protein (GFP), Tw o GFP-mutants with different folding properties, S65T and EGFP, were u sed to produce two recombinant proteins, hCgB-GFP(S65T) and hCgB-EGFP, respectively. After transient expression only hCgB-EGFP elicited gree n fluorescence in the neuroendocrine cell line PC12. Pulse-chase exper iments with [S-35]sulfate followed by subcellular fractionation showed that hCgB-EGFP was sorted with high efficiency to immature secretory granules (ISG), Confocal microscopy revealed that fluorescent hCgB-EGF P colocalized largely with synaptotagmin, a membrane marker of secreto ry granules and synaptic-like microvesicles, and significantly with en dogenous rat chromogranin B (rCgB), a soluble marker of secretory gran ules, Upon stimulation of transfected cells with 5 mM Ba2+ or by depol arization with 50 mM K+ hCgB-EGFP underwent regulated exocytosis. The dynamics of green fluorescent secretory granules beneath the plasma me mbrane (PM) of living PC12 cells were visualized by confocal microscop y, The majority of these vesicles did not move within 8.5 sec as if th ey were docked, In contrast, in NGF-induced cells most of the secretor y granules beneath the somatic PM moved within the same time period wh ereas only little movement was observed in the neurites. These finding s indicate that in differentiated PC12 cells the majority of the docki ng zones are not in the soma but are distributed along the neurites, I n conclusion, the fusion protein hCgB-EGFP provides a powerful tool to study in real time vesicular traffic in the regulated pathway of prot ein secretion.