MagIC, a genetically encoded fluorescent indicator for monitoring cellular Mg2+ using a non-Förster resonance energy transfer ratiometric imaging approach

Abstract

Intracellular Mg(2+) roles are commensurate with its abundance in the cell cytoplasm. However, little is known about Mg(2+) subcellular dynamics, primarily due to the lack of suitable Mg(2+)-selective tools to monitor this ion in intracellular compartments. To cope with this lack, we developed a Mg(2+)-sensitive indicator--MagIC (indicator for Magnesium Imaging in Cell)--composed of a functionalized yellow fluorescent protein (FP) variant fused to a red-emitting FP serving as a reference, thus allowing ratiometric imaging of Mg(2+) MagIC expressed in mammalian cells is homogeneously distributed between the cytosol and nucleus but its fusion with appropriate targeting sequences redirects it to mitochondria or the endoplasmic reticulum. MagIC shows little interference by intracellular Ca(2+) [Kd (Mg(2+)) = 5.1 mM; Kd (Ca(2+)) = 4.8 mM] and its kinetic properties (k(off) = 84 s(-1)) approach those of indicator dyes. With MagIC, as reported previously, we also observed a cytosolic Mg(2+) increase provoked by application of 50 mM MgCl2 in the medium. This effect is, however, mimicked by 75 mM KCl or 150 mM D-sorbitol addition, indicating that it is a response to the associated hyperosmotic shock and not to Mg(2+) itself. Our results confirm the functionality of MagIC as a useful tool for the long-awaited possibility of prolonged and organelle-specific monitoring of cellular Mg(2+).

Visualization of long-term Mg2+dynamics in apoptotic cells using a novel targetable fluorescent probe

“…To improve the spatial and temporal resolution of Mg 2+ imaging, various Mg 2+ probes have been developed. Recently, genetically encoded fluorescent protein-based Mg 2+ sensors, MagFRET 14 and MagIC, 15 have been reported. These probes were easily targeted to various intracellular compartments by adding a localization signal peptide, and are likely to be capable of long-term detection.…”

In vivostudy of gene expression with an enhanced dual-color fluorescent transcriptional timer

“…4a). Previously, ratiometric imaging of FPs based on their different properties, such as maturation time and sensitivity to specific ions, has been used successfully to measure protein life-time 31 , pH changes 33,34 , and Mg 2+ concentration 35 . We reason that dynamic transcriptional activities might also be detected similarly using the ratio between dGFP and RFP.…”

Design and development of genetically encoded fluorescent sensors to monitor intracellular chemical and physical parameters

“…The authors state that the precise geometric properties generated by these substitutions are responsible for a Ca 2+ chelation near the chromophore. Based on the same strategy, Koldenkova and colleagues screened the mutations in cpVenus to selectively sense Mg 2+ or Ca 2+ ions and successfully found the right mutations (Koldenkova et al 2015 ). While the intracellular concentration of Mg 2+ in mammalian cells is in the range of 15–20 mM, Ca 2+ concentrations are <1 mM.…”