AUTHOR=Asano Toshifumi , Igarashi Hiroyuki , Ishizuka Toru , Yawo Hiromu TITLE=Organelle Optogenetics: Direct Manipulation of Intracellular Ca2+ Dynamics by Light JOURNAL=Frontiers in Neuroscience VOLUME=12 YEAR=2018 URL=https://www.frontiersin.org/journals/neuroscience/articles/10.3389/fnins.2018.00561 DOI=10.3389/fnins.2018.00561 ISSN=1662-453X ABSTRACT=

As one of the ubiquitous second messengers, the intracellular Ca²⁺, has been revealed to be a pivotal regulator of various cellular functions. Two major sources are involved in the initiation of Ca²⁺-dependent signals: influx from the extracellular space and release from the intracellular Ca²⁺ stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR). To manipulate the Ca²⁺ release from the stores under high spatiotemporal precision, we established a new method termed “organelle optogenetics.” That is, one of the light-sensitive cation channels (channelrhodopsin-green receiver, ChRGR), which is Ca²⁺-permeable, was specifically targeted to the ER/SR. The expression specificity as well as the functional operation of the ER/SR-targeted ChRGR (ChRGR_ER) was evaluated using mouse skeletal myoblasts (C2C12): (1) the ChRGR_ER co-localized with the ER-marker KDEL; (2) no membrane current was generated by light under whole-cell clamp of cells expressing ChRGR_ER; (3) an increase of fluorometric Ca²⁺ was evoked by the optical stimulation (OS) in the cells expressing ChRGR_ER in a manner independent on the extracellular Ca²⁺ concentration ([Ca²⁺]_o); (4) the ΔF/F₀ was sensitive to the inhibitor of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and (5) the store-operated Ca²⁺ entry (SOCE) was induced by the OS in the ChRGR_ER-expressing cells. Our organelle optogenetics effectively manipulated the ER/SR to release Ca²⁺ from intracellular stores. The use of organelle optogenetics would reveal the neuroscientific significance of intracellular Ca²⁺ dynamics under spatiotemporal precision.