3 research outputs found
GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles
ProducciĂłn CientĂficaGenetically encoded calcium indicators allow monitoring subcellular
Ca2+ 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 Ca2+
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 Mg2+,
tunable Ca2+ 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 Ca2+ indicators for organelles and becomes a valuable tool
for in vivo Ca2+ imaging applications
GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles
Genetically encoded calcium indicators allow monitoring subcellular Ca2+ 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 Ca2+ 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 Mg2+, tunable Ca2+ 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 Ca2+ indicators for organelles and becomes a valuable tool for in vivo Ca2+ imaging applications.This work was supported by grants from the European Research Area Net (ERA-Net) program, the Spanish Ministerio de EconomĂa y Competitividad (SAF2008-03175-E, BFU2010-17379), and the Instituto de Salud Carlos III (RD06/0010/0000).Peer Reviewe