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.
Highlights
As a ubiquitous intracellular messenger, Ca2+ has essential physiological roles in a variety of cellular processes including fertilization and development, muscle contraction and neurotransmitter release [1]
As Forster resonance energy transfer (FRET) is strongly dependent on the distance between donor and acceptor fluorophores, the conformational change associated with the Ca2+ -dependent interaction of CaM and M13 leads to a change in FRET efficiency and a ratiometric change in the fluorescence signal [2]
In an effort to circumvent these problems, we explored the introduction of orthogonal CaM–M13 pairs in RGECO1 and R-GECO1.2 by mutagenesis of residues involved in interactions at the CaM–M13 interface
Summary
As a ubiquitous intracellular messenger, Ca2+ has essential physiological roles in a variety of cellular processes including fertilization and development, muscle contraction and neurotransmitter release [1]. When expressed in HeLa cells, ER-LAR-GECO1 showed significantly higher fluorescence intensity compared with ER-R-GECO1 and ER-R-GECO1.2 (B, D and F) Effects of depleting the ER of Ca2 + by addition of thapsigargin (10 μM) on the fluorescence intensities of ER-LAR-GECO1 (red line) and CatchER (green line) co-expressed in a HeLa cell, a HEK-293 cell and a U2-OS cell as shown in (A), (C) and (E), respectively.
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