title: GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles.
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.
authors: Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, 47003 Valladolid, Spain.
authors: Arancha
authors: A
authors: Rodriguez-Garcia
authors: Jonathan
authors: J
authors: Rojo-Ruiz
authors: Paloma
authors: P
authors: Navas-Navarro
authors: Francisco Javier
authors: FJ
authors: Aulestia
authors: Sonia
authors: S
authors: Gallego-Sandin
authors: Javier
authors: J
authors: Garcia-Sancho
authors: Maria Teresa
authors: MT
authors: Alonso
mesh_terms: D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D057054:Molecular Imaging / Q000379:methods*; D015388:Organelles / Q000737:chemistry*
chemical_list: D049452:Green Fluorescent Proteins; D000331:Aequorin; D002118:Calcium
keywords: Golgi apparatus; calcium signalling; hippocampus; motor neuron