Dissection of local Ca(2+) signals inside cytosol by ER-targeted Ca(2+) indicator.
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.
Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute (BSI), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Fumihiro
F
Niwa
Division of Biological Science, Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi 464-8602, Japan.
Shigeo
S
Sakuragi
Division of Biological Science, Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi 464-8602, Japan.
Ayana
A
Kobayashi
Division of Biological Science, Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi 464-8602, Japan.
Shin
S
Takagi
Division of Biological Science, Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi 464-8602, Japan.
Yoichi
Y
Oda
Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute (BSI), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Division of Biological Science, Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi 464-8602, Japan; Nagoya Research Center for Brain & Neural Circuits, Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi 464-8602, Japan; Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: hbannai@brain.riken.jp.
Hiroko
H
Bannai
Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute (BSI), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Electronic address: mikosiba@brain.riken.jp.
Katsuhiko
K
Mikoshiba
D000818:Animals; D030801:Animals, Genetically Modified; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D019556:COS Cells; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D059008:Time-Lapse Imaging / Q000379:methods
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium
Astrocyte; C. elegans; Endoplasmic reticulum; Genetically encoded Ca(2+) indicator; Local Ca(2+)