GAP, an aequorin-based fluorescent indicator for imaging Ca ²⁺ 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.calcium signalling | Golgi apparatus | hippocampus | motor neuron

GFP-Aequorin Protein Sensor for Ex Vivo and In Vivo Imaging of Ca 2+ Dynamics in High-Ca 2+ Organelles

“…In summary, the optimized Ca 2+ sensors represent a substantial improvement over previous GAPs due to its optimal K D for measurements in high [Ca 2+ ] compartments, its ratiometric measurements, large dynamic range, and uncomplicated calibration (Rodriguez-Garcia et al, 2014). Even more relevant is its bioorthogonal nature, which facilitates robust functional expression of the indicator with no apparent off-target effects.…”

“…[Ca 2+ ] C was also measured using Fura-2 ( Figure S2B) as described previously (Rodriguez-Garcia et al, 2014). Astrocyte imaging was performed similarly.…”

“…We have recently described a new family of fluorescent Ca 2+ sensors named GAP (GFP-aequorin protein) (Rodriguez-Garcia et al, 2014). A unique feature of this family is the use of aequorin instead of calmodulin or troponin-C to provide the Ca 2+ -binding sites.…”

Fluorescent Protein–photoprotein Fusions and Their Applications in Calcium Imaging

“…This novel class of GECI deserves a special comment. They are molecular fusions of a particular GFP mutant (GFPuv) and aequorin, able to function either as a luminescent or fluorescent biosensors for Ca 2+ , depending on whether apoaequorin is reconstituted with coelenterazine or not.…”

Endoplasmic reticulum stress in insulin resistance and diabetes

“…This large Ca 2+ gradient between cytoplasm and the ER requires both the action of sarcoendoplasmic reticulum Ca 2+ ATPase (SERCA pump) and a reduced activity of leak and release Ca 2+ channels present in the membrane of ER. Using ratiometric genetically encoded luminal ER Ca 2+ indicators has been observed a large variability in the resting [Ca 2+ ] L [5,6]. Since this is more evident in the upper end of the [Ca 2+ ] L determinations, it has been argued that this is artificial due to saturation of the ratiometric indicators [7].…”