Development of multi-scale and functional imaging technique to quantify the fluctuation in intercellular signaling

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To better understand the principle in self-organized pattern formation, it is important to measure fluctuating input/output-relationship of intercellular communication at the onset of pattern formation. Here, we tried to develop ultrasensitive indicators for cAMP, being the major communication signaling molecule. First, we designed the FRET-based cAMP indicator whose dynamic range and Kd was optimized for the extracellular measurement, and successfully generated ultrasensitive cAMP probe whose Kd is around 50 nM. We also developed luminescent-based BRET indicators whose Kd ranges from 50 nM to 3 microM. By utilizing these probes, we can directly measure the pattern of both intra-and intercellular cAMP concentration change which should reveal the input/output-relation during the self-organized pattern formation.