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Fluorescent Cargo Proteins in Pancreatic ß-Cells: Design Determines Secretion Kinetics at Exocytosis

Darren J. Michael ^*, Xuehui Geng , Niamh X. Cawley , Y. Peng Loh , Christopher J. Rhodes , Peter Drain  and Robert H. Chow ^*

^* Department of Physiology and Biophysics, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90089; Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; Section on Cellular Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Pacific Northwest Research Institute, Seattle, Washington 98122

Correspondence: Address reprint requests and inquiries to Robert H. Chow, Tel. 323-442-2901; Fax: 323-442-4466; E-mail: rchow{at}usc.edu.

We compared secretion kinetics for four different fluorescent cargo proteins, each targeted to the lumen of insulin secretory vesicles. Upon stimulation, individual vesicles displayed one of four distinct patterns of fluorescence change: i), disappearance, ii), dimming, iii), transient brightening, or iv), persistent brightening. For each fusion protein, a different pattern of fluorescence change dominated. Furthermore, we demonstrated that the dominant pattern depends upon both i), the specific choice of fluorescent protein, and ii), the sequence of amino acids linking the cargo protein to the fluorescent protein. Thus, in ß-cells, experiments involving fluorescent cargo proteins for the study of exocytosis must be interpreted carefully, as design of a fluorescent cargo protein determines secretion kinetics at exocytosis.