dissection-local-ca2-signals-cultured-cells-membrane-targeted-ca2

Dissection of Local Ca<sup>2+</sup> Signals in Cultured Cells by Membrane-targeted Ca<sup>2+</sup> Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

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RIKEN Center for Brain Science

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.

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

Astrocytes play key roles in the central nervous system and regulate local blood flow and synaptic transmission via intracellular calcium (Ca) signaling. Astrocytic Ca signals are generated by multiple pathways: Ca release from the endoplasmic reticulum (ER) via the inositol 1, 4, 5-trisphosphate receptor (IPR) and Ca influx through various Ca channels on the plasma membrane. However, the Ca channels involved in astrocytic Ca homeostasis or signaling have not been fully characterized. Here, we demonstrate that spontaneous astrocytic Ca transients in cultured hippocampal astrocytes were induced by cooperation between the Ca release from the ER and the Ca influx through store-operated calcium channels (SOCCs) on the plasma membrane. Ca imaging with plasma membrane targeted GCaMP6f revealed that spontaneous astroglial Ca transients were impaired by pharmacological blockade of not only Ca release through IPRs, but also Ca influx through SOCCs. Loss of SOCC activity resulted in the depletion of ER Ca, suggesting that SOCCs are activated without store depletion in hippocampal astrocytes. Our findings indicate that sustained SOCC activity, together with that of the sarco-endoplasmic reticulum Ca-ATPase, contribute to the maintenance of astrocytic Ca store levels, ultimately enabling astrocytic Ca signaling.

Astroglial Ca signaling is generated by the coordination of IPR and store-operated Ca channels.

The fluid mosaic model states that molecules in the plasma membrane can freely undergo lateral diffusion; however, in neurons and glia, specific membrane molecules are concentrated in cellular microdomains to overcome the randomizing effects of free diffusion. This specialized distribution of membrane molecules is crucial for various cell functions; one example is the accumulation of neurotransmitter receptors at the postsynaptic neuronal membrane, which enables efficient synaptic transmission. Quantum dot-single particle tracking (QD-SPT) is a super-resolution imaging technique that uses semiconductor nanocrystal quantum dots as fluorescent probes, and is a powerful tool for analyzing protein and lipid behavior in the plasma membrane. In this article, we review studies implementing QD-SPT in neuroscience research and important data gleaned using this technology. Recent QD-SPT experiments have provided critical insights into the mechanism and physiological relevance of membrane self-organization in neurons and astrocytes in the brain. The mobility of some membrane molecules may become abnormal in cellular models of epilepsy and Alzheimer's disease. Based on these findings, we propose that the behavior of membrane molecules reflects the condition of neurons in pathological disease states.

Hiroko Bannai

Japan Science and Technology Agency

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Dissection of Local Ca²⁺ Signals in Cultured Cells by Membrane-targeted Ca²⁺ Indicators

Hiroko Bannai

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Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Japan Science and Technology Agency

Dissection of Local Ca²⁺ Signals in Cultured Cells by Membrane-targeted Ca²⁺ Indicators