Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels

Abstract

Cytosolic Ca 2+ levels ([Ca 2+ ] c ) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca 2+ ] c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca 2+ ] c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca 2+ ] c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca 2+ -loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca 2+ ] c . Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca 2+ ] c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca 2+ ] c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca 2+ ] c in single cells and animal models.

A Genetically Encoded Bioluminescence Intracellular Nanosensor for Androgen Receptor Activation Monitoring in 3D Cell Models

“…Notably, the reassembly is reversible, thus enabling both protein association and dissociation to be monitored [ 3 ]. NanoBiT has been successfully applied to monitor PPI and detect intracellular calcium levels [ 13 ], endosome disruption [ 14 ], and circulating microRNAs with such a high sensitivity that it could be implemented in smartphone-based assays [ 15 ]. In another configuration, split reporter proteins provide a single-molecule probe to monitor ligand-induced conformational changes in a single molecular backbone [ 16 , 17 , 18 ].…”

A Dual-Purpose Real-Time Indicator and Transcriptional Integrator for Calcium Detection in Living Cells

“…In all cases, the dynamic ranges were insufficient, primarily due to high background in the absence of calcium (Figure S2A-B). We hypothesized that a twocomponent sensor in which calcium drives the reconstitution of a split luciferase, as in Nguyen et al, 2020 33 , may have greater dynamic range due to its requirement for an intermolecular rather than an intramolecular interaction.…”

“…In all cases, the dynamic ranges of these biosensors were insufficient in the context of LuCID, primarily due to high background in the absence of calcium (Figure S2A,B). We hypothesized that a two-component sensor in which calcium drives the reconstitution of a split luciferase, such as in Nguyen et al, 2020, 33 might have a greater dynamic range because of have low affinity for one another (K d = 190 μM) and must be driven together by a protein−protein interaction in order to reconstitute and give bioluminescence. 13 We fused the calcium-binding protein calmodulin (CaM) to LgBiT and its calcium-dependent binding peptide M13 to SmBiT, and measured bioluminescence in HEK293T cells treated with CaCl 2 and ionomycin or left untreated.…”

The Extended N-Terminal Domain Confers Atypical Chemokine Receptor Properties to CXCR3-B

“…CXCR3-driven chemokine-induced calcium flux was assessed using an assay based on nanoluciferase complementation (NanoBiT) and Ca 2+ -dependent calmodulin–MYLK2S protein association ( 42 ). HEK293T cells were plated in a 6-well plate (1 x 10 6 cells per well) and cultured for 24 hours before transfection with CXCR3-A- or CXCR3-B-encoding pIRES vectors and plasmids encoding for calmodulin C-terminally fused to SmBiT and MYLK2S N-terminally fused to LgBiT.…”