Journal article
Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum
The Biochemical journal, Vol.464(1), pp.13-22
11/15/2014
DOI: 10.1042/BJ20140931
PMCID: PMC4214425
PMID: 25164254
Abstract
Ca2+ is a key intermediary in a variety of signalling pathways and undergoes dynamic changes in its cytoplasmic concentration due to release from stores within the endoplasmic reticulum (ER) and influx from the extracellular environment. In addition to regulating cytoplasmic Ca2+ signals, these responses also affect the concentration of Ca2+ within the ER and mitochondria. Single fluorescent protein-based Ca2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concentration of Ca2+ associated with intracellular signalling pathways. Most GCaMP-type indicators have dissociation constants (Kd) for Ca2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca2+], but poorly suited for use in mitochondria and ER where [Ca2+] can reach concentrations of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 μM (LAR-GECO1) and 12 μM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. In addition, we perform two-colour imaging of intracellular Ca2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca2+ indicators enables monitoring of ER and mitochondrial Ca2+ in combination with GFP-based reporters.
Details
- Title: Subtitle
- Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum
- Creators
- Jiahui Wu - Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2David L Prole - †Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, U.KYi Shen - Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2Zhihong Lin - ‡Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA 52242, U.S.AAswini Gnanasekaran - ‡Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA 52242, U.S.AYingjie Liu - §Department of Physiology and Pharmacology, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada, T2N 4N1Lidong Chen - Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2Hang Zhou - Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2S R Wayne ChenYuriy M Usachev - ‡Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA 52242, U.S.AColin W Taylor - †Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, U.KRobert E Campbell - Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2
- Resource Type
- Journal article
- Publication Details
- The Biochemical journal, Vol.464(1), pp.13-22
- DOI
- 10.1042/BJ20140931
- PMID
- 25164254
- PMCID
- PMC4214425
- NLM abbreviation
- Biochem J
- ISSN
- 0264-6021
- eISSN
- 1470-8728
- Publisher
- England
- Grant note
- 101844 / Wellcome Trust NS087068 / NINDS NIH HHS L0000075 / Biotechnology and Biological Sciences Research Council R01 NS087068 / NINDS NIH HHS
- Language
- English
- Date published
- 11/15/2014
- Academic Unit
- Iowa Neuroscience Institute; Anesthesia; Neuroscience and Pharmacology
- Record Identifier
- 9984040379602771
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