Engineering of NEMO as calcium indicators with large dynamics and high sensitivity

Abstract

Genetically encoded calcium indicators (GECIs) are indispensable tools for real-time monitoring of intracellular calcium signals and cellular activities in living organisms. Current GECIs face the challenge of suboptimal peak signal-to-baseline ratio (SBR) with limited resolution for reporting subtle calcium transients. We report herein the development of a suite of calcium sensors, designated NEMO, with fast kinetics and wide dynamic ranges (>100-fold). NEMO indicators report Ca2+ transients with peak SBRs around 20-fold larger than the top-of-the-range GCaMP6 series. NEMO sensors further enable the quantification of absolution calcium concentration with ratiometric or photochromic imaging. Compared with GCaMP6s, NEMOs could detect single action potentials in neurons with a peak SBR two times higher and a median peak SBR four times larger in vivo, thereby outperforming most existing state-of-the-art GECIs. Given their high sensitivity and resolution to report intracellular Ca2+ signals, NEMO sensors may find broad applications in monitoring neuronal activities and other Ca2+-modulated physiological processes in both mammals and plants.

Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants

“…20 By having the biosensor element easily interchangeable, it is easy to update the experimental format as improved biosensors are discovered, such as the new calcium indicator NEMOc. 53 This assay format could be paired with a site-saturation mutagenesis library of a protein capable of modulating intracellular calcium concentrations such as STIM1. Similar biosensor−modulator pairs could easily be tested in the future, such as cyclic adenosine monophosphate (cAMP) indicators 54 to study variants of GNAS 55 in pseudohypoparathyroidism, or pyruvate sensors 56 to study variants in PDHA1 involved in pyruvate dehydrogenase complex deficiency.…”

“…We showed how the second landing pad site could be used to express a cellular biosensor, such as the calcium-sensing jGCaMP7c fluorescent protein . By having the biosensor element easily interchangeable, it is easy to update the experimental format as improved biosensors are discovered, such as the new calcium indicator NEMOc …”

Injectable Fluorescent Neural Interfaces for Cell-Specific Stimulating and Imaging

“…86 The introduction of the jGCaMP8 series has addressed the compromise between resolution and depth penetration inherent in other imaging techniques like FMT and NIRF. Due to its enhanced sensitivity, rapid response, and strong fluorescence, 76,87 the jGCaMP8 series surpasses its predecessors and rivals, including the XCaMP series (Figure 5D). This breakthrough helps balance the need for high-resolution imaging with the depth penetration required, especially given the challenges of brain tissue properties, and the skull's thickness and opacity.…”

Genetically encoded protein sensors for metal ion detection in biological systems: a review and bibliometric analysis

“…NEMOs constructed on the basis of mNeonGreen fluorescent protein can not only have similar dynamic characteristics to GCaMP6, but also its fluorescence intensity, resistance to light quenching, and pH stability have been significantly improved, which provides a new direction for the optimization of genetically encoded Ca 2+ fluorescent protein sensors. 39,61–63…”