GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles

Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2584-9. doi: 10.1073/pnas.1316539111. Epub 2014 Feb 5.

Abstract

Genetically encoded calcium indicators allow monitoring subcellular Ca(2+) signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca(2+) sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg(2+), tunable Ca(2+) affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca(2+) indicators for organelles and becomes a valuable tool for in vivo Ca(2+) imaging applications.

Keywords: Golgi apparatus; calcium signalling; hippocampus; motor neuron.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aequorin / genetics
  • Aequorin / metabolism*
  • Animals
  • Biosensing Techniques / methods*
  • Calcium / analysis*
  • Endoplasmic Reticulum / metabolism
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Mice
  • Mice, Transgenic
  • Molecular Imaging / methods*
  • Organelles / chemistry*

Substances

  • Green Fluorescent Proteins
  • Aequorin
  • Calcium