SECRETORY-GRANULE DYNAMICS VISUALIZED IN-VIVO WITH A PHOGRIN GREEN FLUORESCENT PROTEIN CHIMERA

To image the behaviour in real time of single secretory granules in ne uroendocrine cells we have expressed cDNA encoding a fusion construct between the dense-core secretory-granule-membrane glycoprotein, phogri n (phosphatase on the granule of insulinoma cells), and enhanced green fluorescent protein (EGFP). Expressed in INS-1 beta-cells and pheochr omocytoma PC12 cells, the chimaera was localized efficiently (up to 95 %) to dense-core secretory granules (diameter 200-1000 nm)? identifie d by co-immunolocalization with anti-(pro-)insulin antibodies in INS-I cells and dopamine beta-hydroxylase in PC12 cells. Using laser-scanni ng confocal microscopy and digital image analysis, we have used this c himaera to monitor the effects of secretagogues on the dynamics of sec retory granules in single living cells. In unstimulated INS-1 beta-cel ls, granule movement was confined to oscillatory movement (dithering) with period of oscillation 5-10 s and mean displacement <1 mu m. Both elevated glucose concentrations (30 mM), and depolarization of the pla sma membrane with K+, provoked large (5-10 mu m) saltatory excursions of granules across the cell, which were never observed in cells mainta ined at low glucose concentration. By contrast, long excursions of gra nules occurred in PC 12 cells without stimulation, and occurred predom inantly from the cell body towards the cell periphery and neurite exte nsions. Purinergic-receptor activation with ATP provoked granule movem ent towards the membrane of PC12 cells, resulting in the transfer of f luorescence to the plasma membrane consistent with fusion of the granu le and diffusion of the chimaera in the plasma membrane. These results illustrate the potential use of phogrin-EGFP chimeras in the study of secretory-granule dynamics, the regulation of granule-cytoskeletal in teractions and the trafficking of a granule-specific transmembrane pro tein during the cycle of exocytosis and endocytosis.