Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum

• DOI: 10.1042/bj20140931
• Publication Date: 2014-08-29T08:31:25Z
• Authors: Jiahui Wu; David L. Prole; Yi Shen; Zhihong Lin; Aswini Gnanasekaran; Yingjie Liu; Lidong Chen; Hang Zhou; S. R. Wayne Chen; Yuriy M. Usachev; Colin W. Taylor; Robert E. Campbell
• Keywords: Calcium Signaling; Live cell imaging

Ca2+ is a key intermediary in variety of signalling pathways and undergoes dynamic changes its cytoplasmic concentration due to release from stores within the endoplasmic reticulum (ER) influx extracellular environment. In addition regulating signals, these responses also affect ER mitochondria. Single fluorescent protein-based indicators, such as GCaMP series based on GFP, are powerful tools for imaging associated with intracellular pathways. Most GCaMP-type indicators have dissociation constants (Kd) high nanomolar low micromolar range therefore optimal measuring [Ca2+], but poorly suited use mitochondria where [Ca2+] can reach concentrations several hundred micromolar. We now report low-affinity red genetically encoded optical (LAR-GECO), engineered Kd values 24 μM (LAR-GECO1) 12 (LAR-GECO1.2). demonstrate that be used image mitochondrial dynamics cell types. addition, we perform two-colour cells expressing both ER-targeted LAR-GECO1. The development intensiometric enables monitoring combination GFP-based reporters.