Dissection of local Ca²⁺ signals inside cytosol by ER-targeted Ca²⁺ indicator

Calcium (Ca²⁺) is a versatile intracellular second messenger that operates in various signaling pathways leading to multiple biological outputs. The diversity of spatiotemporal patterns of Ca²⁺ signals, generated by the coordination of Ca²⁺ influx from the extracellular space and Ca²⁺ release from the intracellular Ca²⁺ store the endoplasmic reticulum (ER), is considered to underlie the diversity of biological outputs caused by a single signaling molecule. However, such Ca²⁺ signaling diversity has not been well described because of technical limitations. Here, we describe a new method to report Ca²⁺ signals at subcellular resolution. We report that OER-GCaMP6f, a genetically encoded Ca²⁺ indicator (GECI) targeted to the outer ER membrane, can monitor Ca²⁺ release from the ER at higher spatiotemporal resolution than conventional GCaMP6f. OER-GCaMP6f was used for in vivo Ca²⁺ imaging of C. elegans. We also found that the spontaneous Ca²⁺ elevation in cultured astrocytes reported by OER-GCaMP6f showed a distinct spatiotemporal pattern from that monitored by plasma membrane-targeted GCaMP6f (Lck-GCaMP6f); less frequent Ca²⁺ signal was detected by OER-GCaMP6f, in spite of the fact that Ca²⁺ release from the ER plays important roles in astrocytes. These findings suggest that targeting of GECIs to the ER outer membrane enables sensitive detection of Ca²⁺ release from the ER at subcellular resolution, avoiding the diffusion of GECI and Ca²⁺. Our results indicate that Ca²⁺ imaging with OER-GCaMP6f in combination with Lck-GCaMP6f can contribute to describing the diversity of Ca²⁺ signals, by enabling dissection of Ca²⁺ signals at subcellular resolution.