Spatiotemporal analyses of cellular signals using new fluorescent indicators

Abstract

Revealing spatiotemporal aspects of cellular signalling molecules is one of the major challenges to current biomedical sciences. We developed new fluorescent indicators to monitor signalling molecules upstream and downstream of intracellular Ca²⁺ signals within living cells. Using the indicators we have clarified important cellular mechanisms, and some of these are listed below. Imaging of inositol 1,4,5-trisphosphate (IP₃) concentration changes within fine dendrites of cerebellar Purkinje cells identified robust cross talk between metabotropic and ionotropic glutamate receptors for the generation of IP₃ signals. Nitric oxide (NO) generation at the parallel fiber-Purkinje cell synapse was imaged and a NO- and frequency-dependent synaptic plasticity was identified. Using organelle-targeted Ca²⁺ indicators, we clarified the involvement of mitochondrial Ca²⁺ handling in the generation of Ca²⁺ oscillations. We generated a fluorescence energy transfer-based indicator of myosin light chain phosphorylation, which can monitor the phosphorylation state of myosin light chain within intact cells. These results demonstrate the power of fluorescent indicators in physiological studies of a variety of cell functions. These new methods may also shed light on the regulatory mechanism of smooth muscle contraction. [Jpn J Physiol 55 Suppl:S15 (2005)]