Spatiotemporal mapping of multidimensional chemokine dynamics by genetically encoded fluorescent sensors.
Chemokines are a class of protein-based extracellular signals that serve as guidance cues for cell migration, thereby playing central roles in development and immune homeostasis. Dynamic patterns of chemokines are required to faithfully translate the progressive changes in local environments, yet the precise organization of chemokine signaling in time and space remains largely uncharacterized due to limited techniques for tracking them in vivo. Using CXCR3 as a prototype, we engineer a genetically encoded fluorescent sensor for its chemokine ligands CXCL9-11 by inserting a circular-permutated fluorescent protein into the CXCR3 receptor. The new sensor GRAB-LoX3-1.0 exhibits a high signal-to-noise ratio in terms of fluorescence response and nanomolar affinity for corresponding chemokines, and is equipped with tens of milliseconds of temporal resolution and submicrometer spatial resolution. By applying LoX3-1.0, we monitor the temporal progression of extracellular chemokine patterns that are shaped by distinct inflammatory states, and quantitatively reveal the multidimensional properties of chemokine signaling as well as its potential organizational principles. Furthermore, we achieve direct visualization of the micrometer-scale CXCL10 gradient and its temporal evolution around blood vessels in the native brain environment after inflammation. Transgenic expression of LoX3-1.0 also enables the precise recording of injury-evoked CXCL10 dynamics in the peripheral skin of mice in vivo. In summary, newly engineered LoX3-1.0 sensor faithfully reports the spatiotemporal properties of chemokine dynamics, which reveals diverse and context-specific patterns as the basis of their function.