Dissection of local Ca(2+) signals inside cytosol by ER-targeted Ca(2+) indicator

• Published in: Biochemical and biophysical research communications 2016-10, Vol.479 (1), p.67-73
• Main Authors: Niwa, Fumihiro, Sakuragi, Shigeo, Kobayashi, Ayana, Takagi, Shin, Oda, Yoichi, Bannai, Hiroko, Mikoshiba, Katsuhiko
• Format: Article
• Language: English
• Subjects: Animals; Animals, Genetically Modified; Astrocytes - cytology; Astrocytes - metabolism; Caenorhabditis elegans - genetics; Caenorhabditis elegans - metabolism; Calcium - metabolism; Calcium Signaling; Cell Membrane - metabolism; Cells, Cultured; Cercopithecus aethiops; COS Cells; Cytosol - metabolism; Endoplasmic Reticulum - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HeLa Cells; Humans; Microscopy, Confocal; Rats, Wistar; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Time-Lapse Imaging - methods
• ISSN: 1090-2104
• DOI: 10.1016/j.bbrc.2016.09.034

Calcium (Ca(2+)) is a versatile intracellular second messenger that operates in various signaling pathways leading to multiple biological outputs. The diversity of spatiotemporal patterns of Ca(2+) signals, generated by the coordination of Ca(2+) influx from the extracellular space and Ca(2+) release from the intracellular Ca(2+) store the endoplasmic reticulum (ER), is considered to underlie the diversity of biological outputs caused by a single signaling molecule. However, such Ca(2+) signaling diversity has not been well described because of technical limitations. Here, we describe a new method to report Ca(2+) signals at subcellular resolution. We report that OER-GCaMP6f, a genetically encoded Ca(2+) indicator (GECI) targeted to the outer ER membrane, can monitor Ca(2+) release from the ER at higher spatiotemporal resolution than conventional GCaMP6f. OER-GCaMP6f was used for in vivo Ca(2+) imaging of C. elegans. We also found that the spontaneous Ca(2+) 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 Ca(2+) signal was detected by OER-GCaMP6f, in spite of the fact that Ca(2+) 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 Ca(2+) release from the ER at subcellular resolution, avoiding the diffusion of GECI and Ca(2+). Our results indicate that Ca(2+) imaging with OER-GCaMP6f in combination with Lck-GCaMP6f can contribute to describing the diversity of Ca(2+) signals, by enabling dissection of Ca(2+) signals at subcellular resolution.