Genetically encoded biosensor for monitoring spatiotemporal dynamics of CCR2 ligands in culture and in vivo

Abstract

Chemokines regulate immune cell migration in development, homeostasis and inflammation, but the precise spatiotemporal pattern of chemokine release in vivo remains elusive due to the constraints of existing detection methodologies. Here, we report the engineering and characterization of a genetically encoded green fluorescent chemokine sensor, named CRAFi-CCR2, which utilizes the CCR2 receptor as a sensing moiety. In astrocytes, hCRAFi-CCR2, derived from the human CCR2B receptor, exhibited ~300% increase in fluorescence in response to mCCL2, with nanomolar affinity (2.5 nM). Activation of hCRAFi-CCR2 did not affect downstream signaling pathways, such as calcium mobilization and receptor internalization. Using this sensor, we performed 17-20 h of real-time imaging to observe endogenous mCCL2 release under inflammatory conditions, both in cell culture and in mice. In mouse brain, we observed spatial heterogeneity of CCL2 signal response on a scale of about 20-50 µm, highlighting the complexity of the immune system's spatiotemporal signaling.