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Review
. 2008 Nov;46(3):152-9.
doi: 10.1016/j.ymeth.2008.09.015. Epub 2008 Oct 9.

Measuring calcium dynamics in living cells with genetically encodable calcium indicators

Janet E McCombs  1 , Amy E Palmer
Affiliations
Review

Measuring calcium dynamics in living cells with genetically encodable calcium indicators

Janet E McCombs et al. Methods. 2008 Nov.

Abstract

Genetically encoded calcium indicators (GECIs) allow researchers to measure calcium dynamics in specific _targeted locations within living cells. Such indicators enable dissection of the spatial and temporal control of calcium signaling processes. Here we review recent progress in the development of GECIs, highlighting which indicators are most appropriate for measuring calcium in specific organelles and localized domains in mammalian tissue culture cells. An overview of recent approaches that have been undertaken to ensure that the GECIs are minimally perturbed by the cellular environment is provided. Additionally, the procedures for introducing GECIs into mammalian cells, conducting calcium imaging experiments, and analyzing data are discussed. Because organelle-_targeted indicators often pose an additional challenge, we underscore strategies for calibrating GECIs in these locations.

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Figures

Figure 1
Figure 1
Models of the three classes of GECIs. (a) The aequorin photoprotein is shown in complex with coelenterazine. Upon binding of Ca2+, the aequorin undergoes a conformational change, releasing coelenteramide and emitting blue light. (b) Single FP sensors employing the Ca2+-responsive element CaM and a CaM binding peptide attached to a circularly permutated FP. On binding Ca2+, CaM executes a conformational change, interacting with the peptide and altering the protonation state of the chromophore, thus changing the fluorescence intensity of the protein. Note the Case sensors are built from a cpYFP with a T203F and a few other mutations. For details see reference (13). (c) Grafted sensors utilizing EF hands or portions of CaM inserted into a fluorescent protein. Binding of Ca2+ causes a change in protein conformation and a shift in the protonation state of the chromophore. (d) FRET-based sensors having a Ca2+ binding domain located between two flurophorescent proteins. As Ca2+ binds, the Ca2+ binding domain undergoes a conformational change, interacting with its binding peptide. This brings the two FPs closer together, increasing the efficiency of FRET. Below each model are maps for the various available families of GECIs.
Figure 2
Figure 2
Fluorescence images of localized GECIs. (A) nucleus, (B) Golgi, (C) mitochondria, (D) Plasma membrane, and (E) ER. Localization was accomplished by addition of a nuclear localization sequence (PKKKRKVEDA at the C-terminus); fusion to the 81-amino acids at N-terminus of human galactosyltransferase type II; incorporation of 4 repeats of the cytochrome C oxidase signal sequence at the N-terminus; addition of the lyn kinase myristoylation-palmitoylation sequence (MGCIKSKRKDNLNDDGVDMKT) at the N-terminus; and incorporation of both the calreticulin signal sequence at the N-terminus and a KDEL ER-retention tag at the C-terminus.
Figure 3
Figure 3
Representative examples of experiments employing cameleon-type GECIs. Plots are given as FRET ratio versus time for (a) the ER-_targeted cameleon D1ER and (b) the mitochondria-_targeted cameleon 4mtD3cpv. Changes in Ca2+ over time are easily observable upon addition of 4µM thapsigargin and 5µM EGTA/5mM ionomycin for D1ER (a) or 5µM ATP, 5µM ionomycin/5mM EGTA, and 5µM ionomycin/5mM Ca2+ for 4mtD3cpv(b).
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References

    1. Berridge MJ, Bootman MD, Roderick HL. Nature Molecular Cell Biology. 2003;4:517–529. - PubMed
    1. Berridge MJ, Lipp P, Bootman MD. Nature Molecular Cell Biology. 2000;1:11–21. - PubMed
    1. Rizzuto R, Brini M, Pozzan T. Methods Cell Biol. 1994;40:339–358. - PubMed
    1. Robert V, Pinton P, Tosello V, Rizzuto R, Pozzan T. Methods Enzymol. 2000;327:440–456. - PubMed
    1. Brini M, Pinton P, Pozzan T, Rizzuto R. Microscopy Research and Technique. 1999;46:380–389. - PubMed

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