Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis

Biophys J. 2004 Dec;87(6):L03-5. doi: 10.1529/biophysj.104.052175. Epub 2004 Oct 29.

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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Artifacts
  • Carrier Proteins / metabolism*
  • Carrier Proteins / ultrastructure
  • Cells, Cultured
  • Exocytosis / physiology*
  • Fluorescent Dyes / metabolism
  • Insulin / metabolism*
  • Insulin Secretion
  • Islets of Langerhans / cytology
  • Islets of Langerhans / metabolism*
  • Microscopy, Fluorescence / methods*
  • Protein Engineering / methods
  • Rats
  • Recombinant Fusion Proteins / metabolism
  • Secretory Vesicles / metabolism*
  • Secretory Vesicles / ultrastructure

Substances

  • Carrier Proteins
  • Fluorescent Dyes
  • Insulin
  • Recombinant Fusion Proteins