Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis.
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.
Michael
Darren J
DJ
Department of Physiology and Biophysics, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90089, USA.
Geng
Xuehui
X
Cawley
Niamh X
NX
Loh
Y Peng
YP
Rhodes
Christopher J
CJ
Drain
Peter
P
Chow
Robert H
RH
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
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D007328:Insulin; D011993:Recombinant Fusion Proteins