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.
|ジャーナル||Biochemical and Biophysical Research Communications|
|出版ステータス||Published - 2016 10 7|
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