Targeting of green fluorescent protein to neuroendocrine secretory granules: A new tool for real time studies of regulated protein secretion

Christoph Kaether, Thorsten Salm, Michael Glombik, Wolfhard Almers, Hans Hermann Gerdes

Research output: Contribution to journalArticle

75 Scopus citations

Abstract

Human chromogranin B (hCgB), a soluble marker protein of neuroendocrine secretory granules, was fused to green fluorescent protein (GFP). Two GFP-mutants with different folding properties, S65T and EGFP, were used to produce two recombinant proteins, hCgB-GFP(S65T) and hCgB-EGFP, respectively. After transient expression only hCgB EGFP elicited green fluorescence in the neuroendocrine cell line PC12. Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that hCgB-EGFP was sorted with high efficiency to immature secretory granules (ISG). Confocal microscopy revealed that fluorescent hCgB-EGFP colocalized largely with synaptotagmin, a membrane marker of secretory granules and synaptic-like microvesicles, and significantly with endogenous rat chromogranin B (rCgB), a soluble marker of secretory granules. Upon stimulation of transfected cells with 5 mM Ba2+ or by depolarization with 50 mM K+ hCgB-EGFP underwent regulated exocytosis. The dynamics of green fluorescent secretory granules beneath the plasma membrane (PM) of living PC12 cells were visualized by confocal microscopy. The majority of these vesicles did not move within 8.5 sec as if they were docked. In contrast, in NGF-induced cells most of the secretory granules beneath the somatic PM moved within the same time period whereas only little movement was observed in the neurites. These findings indicate that in differentiated PC12 cells the majority of the docking zones are not in the soma but are distributed along the neurites. In conclusion, the fusion protein hCgB-EGFP provides a powerful tool to study in real time vesicular traffic in the regulated pathway of protein secretion.

Original languageEnglish (US)
Pages (from-to)133-142
Number of pages10
JournalEuropean Journal of Cell Biology
Volume74
Issue number2
StatePublished - Oct 1997

Keywords

  • Chromogranin B
  • GFP
  • Real time
  • Regulated secretion
  • Secretory granules

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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