RRC ID 52003
Author Niwa F, Sakuragi S, Kobayashi A, Takagi S, Oda Y, Bannai H, Mikoshiba K.
Title Dissection of local Ca(2+) signals inside cytosol by ER-targeted Ca(2+) indicator.
Journal Biochem Biophys Res Commun
Abstract 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.
Volume 479(1)
Pages 67-73
Published 2016-10-7
DOI 10.1016/j.bbrc.2016.09.034
PII S0006-291X(16)31485-1
PMID 27616195
MeSH Animals Animals, Genetically Modified Astrocytes / cytology Astrocytes / metabolism COS Cells Caenorhabditis elegans / genetics Caenorhabditis elegans / metabolism Calcium / metabolism* Calcium Signaling* Cell Membrane / metabolism Cells, Cultured Chlorocebus aethiops 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
IF 2.985
Times Cited 8
Human and Animal Cells COS-7(RCB0539) HeLa(RCB0007)