ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes

Surya P. Aryal, Mengfan Xia, Ebubechi Adindu, Caroline Davis, Pavel I. Ortinski, Christopher I. Richards

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Ca2+ is a major second messenger involved in cellular and subcellular signaling in a wide range of cells, including astrocytes, which use calcium ions to communicate with other cells in the brain. Even though a variety of genetically encoded Ca2+ indicators have been developed to study astrocyte calcium signaling, understanding the dynamics of endoplasmic reticulum calcium signaling is greatly limited by the currently available tools. To address this, we developed an endoplasmic reticulum-targeted calcium indicator, ER-GCaMP6f, which is anchored to the cytosolic side of the organelle and measures signaling that occurs in close proximity to the endoplasmic reticulum of astrocytes. Using a combination of confocal and super-resolution microscopy techniques, we demonstrate the localization of the indicator in the endoplasmic reticulum in both cell soma and processes of astrocytes. Combining ER-GCaMP6f with total internal reflection fluorescence microscopy, we show that Ca2+ fluctuations in small astrocytic processes can be detected, which are otherwise not observable with existing indicators and standard wide-field and confocal techniques. We also compared the ER-GCaMP6f indicator against currently used plasma membrane-tethered and cytosolic GCaMP6f indicators. ER-GCaMP6f identifies dynamics in calcium signaling of endoplasmic reticulum resident receptors that are missed by plasma membrane-anchored indicators. We also generated an adeno-associated virus (AAV5) and demonstrate that ER-GCaMP6f can be expressed in vivo and by measured calcium activity in brain slices. ER-GCaMP6f provides a powerful tool to study calcium signaling in close proximity to the endoplasmic reticulum in astrocyte cell soma and processes both in culture and in brain slices.

Original languageEnglish
Pages (from-to)2099-2108
Number of pages10
JournalAnalytical Chemistry
Volume94
Issue number4
DOIs
StatePublished - Feb 1 2022

Bibliographical note

Funding Information:
We acknowledge the Genetic Technologies Core at the Center of Molecular Medicine and Light Microscopy Core, University of Kentucky, for letting us use their facilities. C.I.R. acknowledges support from the National Institute for Health (DA038817). P.I.O. also acknowledges support from the National Institute for Health (R01 DA041513). Schematics were constructed from BioRender ( biorender.com ).

Publisher Copyright:
© 2022 American Chemical Society

ASJC Scopus subject areas

  • Analytical Chemistry

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