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

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.

SUBMITTER: Wu J

PROVIDER: S-EPMC4214425 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundUseful probes of the intracellular environment that target a specific organelle in order to allow direct observation of the changes in these regions is of high current interest. Macrocyclic ligands have already revealed themselves as important selective hosts in some biological applications, forming stable and specific complexes. Therefore, in this paper, several macrocyclic ligands are evaluated as potential molecular probes.MethodologyFour polyammonium macrocycles and one macrotricyclic bearing pyridine and phenanthroline chromophores have been synthesised and evaluated as molecular probes. The cytotoxicity of the compounds has been analyzed using human breast cancer cells (MCF-7), non-cancerous human dermal fibroblasts (NHDF) and human adult dermal skin fibroblasts from a breast cancer patient (P14). All the compounds showed low toxicity at concentrations ranging from 10 nM to 10 µM, except for [32]phen(2)N(4) which proved to be highly cytotoxic for MCF-7 cells. Flow cytometry studies evidenced that the percentage of apoptotic and necrotic MCF-7 and NHDF cells induced by the compounds is considerably low. Also, flow cytometry analysis showed that some compounds seem to modify the mitochondrial membrane potential (MMP) of the cells. Fluorescence microscopy evidenced that compounds easily cross the plasma membrane (5 min) and accumulated into the mitochondria, as confirmed by co-localization with MitoTracker Green™. The fluorescence images also evidenced an intact mitochondria structure after 48 h. Moreover, reticular staining suggestive of endoplasmic reticulum (ER) localization, in addition to the mitochondrial one, has been found by confocal microscopy.ConclusionOur study reveals that compounds Me(2)[28]py(2)N(6), cryptphen, [16]phenN(2), [30]phen(2)N(6), have low toxicity and localize in mitochondria and ER. The ability of these compounds for translocating the cellular membrane (5 min) without special conditioning of the cells or derivatization of the probe, the time-dependent localization (48 h) and the cellular viability provide a proof-of-concept towards their use as promising probes towards biomedical studies.

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

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