Kirkland, T. | bioRxiv Science

bioRxiv2026

Bright calcium-modulated bioluminescent indicators for activity imaging and red photon-assisted synaptic transmission

Bioluminescent calcium sensors have unique uses in neuroscience and neuroengineering, enabling noninvasive imaging of neuronal activity and contactless activation of opsin-expressing neurons. However, the speed, range, and robustness of non-invasive imaging and rewiring or neuronal activity are limited by the radiance and dynamic range of existing bioluminescent calcium sensors. Here, we report the stepwise engineering of improved cyan-excitable red fluorescent proteins (mCyRFP4 and dCyRFP4), an improved red bioluminescent protein based on NanoLuc and CyRFP4 (Antares3), and an improved bioluminescent calcium sensor based on Antares3 (CaMBI3). Antares3 is 3-fold brighter than its predecessor, while CaMBI3 responds with an overall dynamic range of 24-fold, enabling high-sensitivity detection of calcium dynamics in muscle and neuronal tissues in vivo. Finally, a CaMBI3 variant and a red-shifted opsin ChRmine enabled red photon-assisted synaptic transmission in C. elegans. CaMBI3 thus facilitates genetically targeted non-invasive imaging and rewiring of neural activity in living animals.

neuroscience↗

bioRxiv2022

Kinase-modulated bioluminescent indicators enable noninvasive imaging of drug activity in the brain

Aberrant kinase activity contributes to the pathogenesis of brain cancers, neurodegeneration, and neuropsychiatric diseases, but identifying kinase inhibitors that function in the brain is challenging. Drug levels in blood do not predict efficacy in the brain because the blood-brain barrier prevents entry of most compounds. Rather, assessing kinase inhibition in the brain requires tissue dissection and biochemical analysis, a time-consuming and resource-intensive process. Here, we report kinase-modulated bioluminescent indicators (KiMBIs) for non-invasive longitudinal imaging of drug activity in the brain based on a recently optimized luciferase-luciferin system. We develop an ERK KiMBI to report inhibitors of the Ras-Raf-MEK-ERK pathway, for which no bioluminescent indicators previously existed. ERK KiMBI discriminates between brain-penetrant and non-penetrant MEK inhibitors, reveals blood-tumor barrier leakiness in xenograft models, and reports MEK inhibitor pharmacodynamics in native brain tissues and intracranial xenografts. Finally, we use ERK KiMBI to screen ERK inhibitors for brain efficacy, identifying temuterkib as a promising brain-active ERK inhibitor, a result not predicted from chemical characteristics alone. Thus, KiMBIs enable the rapid identification and pharmacodynamic characterization of kinase inhibitors suitable for treating brain diseases.

pharmacology and toxicology↗