Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis

PLoS One. 2015 Jun 19;10(6):e0127801. doi: 10.1371/journal.pone.0127801. eCollection 2015.


Fluorescent proteins (FPs) have proven to be valuable tools for high-resolution imaging studies of vesicle transport processes, including exo- and endocytosis. Since the pH of the vesicle lumen changes between acidic and neutral during these events, pH-sensitive FPs with near neutral pKa, such as pHluorin, are particularly useful. FPs with pKa>6 are readily available in the green spectrum, while red-emitting pH-sensitive FPs are rare and often not well characterized as reporters of exo- or endocytosis. Here we tested a panel of ten orange/red and two green FPs in fusions with neuropeptide Y (NPY) for use as secreted vesicle marker and reporter of dense core granule exocytosis and release. We report relative brightness, bleaching rate, targeting accuracy, sensitivity to vesicle pH, and their performance in detecting exocytosis in live cells. Tandem dimer (td)-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP. Single exocytosis events were readily observed, which allowed measurements of fusion pore lifetime and the dynamics of the exocytosis protein syntaxin at the release site during membrane fusion and cargo release.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers
  • Cell Line
  • Cytoplasmic Granules / metabolism*
  • Exocytosis*
  • Hydrogen-Ion Concentration
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Microscopy, Fluorescence
  • Protein Stability
  • Recombinant Fusion Proteins
  • Secretory Vesicles / metabolism*


  • Biomarkers
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • red fluorescent protein

Grant support

The work was supported by the Swedish Research Council, Swedish Diabetes Society, European Foundation for the Study of Diabetes, Diabetes Wellness Network Sweden, Barndiabetesfonden, and the NovoNordisk-, Family Ernfors-, and OE&E Johanssons-foundations.