title: Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis.
abstract: 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 beta-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.
authors.affiliation: Department of Physiology and Biophysics, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90089, USA.
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Michael
authors.forename: Xuehui
authors.initials: X
authors.lastname: Geng
authors.forename: Niamh X
authors.initials: NX
authors.lastname: Cawley
authors.forename: Y Peng
authors.initials: YP
authors.lastname: Loh
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Rhodes
authors.forename: Peter
authors.initials: P
authors.lastname: Drain
authors.forename: Robert H
authors.initials: RH
authors.lastname: Chow
mesh_terms: D000818:Animals; D016477:Artifacts; D002352:Carrier Proteins / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D005089:Exocytosis / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D002352:Carrier Proteins; D005456:Fluorescent Dyes; D007328:Insulin; D011993:Recombinant Fusion Proteins