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

Ca2+ is a key intermediary in a variety of signaling 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 [Ca2+] within the ER and mitochondria. Single fluorescent protein -based Ca2+ indicators, such as the GCaMP series based on the green fluorescent protein (GFP), are powerful tools for imaging changes in the concentration of Ca2+ associated with intracellular signaling pathways. Most GCaMP-type indicators have dissociation constants (Kds) 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 hundreds of micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging, engineereµd 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+ in...

Source: BJ Cell - August 28, 2014 Category: Biochemistry Authors: J Wu, D Lawrence Prole, Y Shen, Z Lin, A Gnanasekaran, Y Liu, L Chen, H Zhou, S Chen, Y M Usachev, C W. Taylor, R E Campbell Tags: BJ ChemBio Source Type: research