We propose to study post-fusional endosomal processing of secretory granules in pituitary lactotrophs, endocrine cells that release the hormone prolactin through granular fusion with the plasma membrane (exocytosis). In pilot studies monitoring exocytosis with the styryl dye FM1-43, we made the unexpected finding that, after endocytic uptake, FM1-43 changes color, shifting from green to red over time. This is the first demonstration of a spectral change in FM dyes in a single cell, and offers exciting new investigative opportunities for these widely used dyes. In the present context, we propose to test the hypothesis that the FM dye color change in lactotrophs reflects endosomal processing of the post-fusional granules. Specifically, we will: a) track the time course of FM1-43 red shift, b) test different colors of FM granules for preferential release, c) test acidification, Zn2+ and Cu2+ effects on FM color and d) immuno-label for proteins required for vesicle re-use.
