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Red and green aequorins for simultaneous monitoring of Ca2+ signals from two different organelles

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Abstract

Simultaneous control of different functions by calcium signals is possible because of subcellular compartmentalization. _targeted chemiluminescent Ca2+ probes, such as aequorins (AEQs) are optimal for detecting signals originating in different subcellular domains, but imaging is difficult because of low photon yield causing poor spatiotemporal resolution. To overcome this problem, we have co-expressed two spectrally distinct AEQs in different subcellular locations within the same cells. Seven chimeric proteins containing either green- or red-emitting AEQs, with different _targeting sequences and Ca2+ affinities, have been designed and tested. We show here evidence for physical and functional independence of the different probes. Cytosolic Ca2+ signals were mirrored in the nucleus, but amplified inside mitochondria and endoplasmic reticulum, and had different time courses in the various locations. Our results demonstrate that these novel tools permit simultaneous and independent monitoring of [Ca2+] in different subcellular domains of the same cell.

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Abbreviations

GFP:

green fluorescent protein

RFP:

red fluorescent protein

mRFP:

monomeric RFP

AEQ:

aequorin

GA:

chimeric GFP-AEQ protein

RA:

chimeric mRFP-AEQ protein

ER:

endoplasmic reticulum

[Ca2+]C :

cytosolic Ca2+ concentration

[Ca2+]N :

nuclear Ca2+ concentration

[Ca2+]ER :

Ca2+ concentration inside ER

[Ca2+]M :

mitochondrial Ca2+ concentration

ψM :

mitochondrial membrane potential

TBH:

2,5-di-tert-butyl-benzohydroquinone

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Acknowledgement

We thank Mr. Jesús Fernández for technical assistance and the Spanish Ministerio de Educación y Ciencia (MEC; BFU2004-02765/BFI, and BFU2005-02078) and Junta de Castilla y León (VA-088/A06) for financial support. IMM and BD held predoctoral fellowships from MEC and Junta de Comunidades de Castilla-La Mancha, respectively.

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Authors and Affiliations

  1. Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, c/Sanz y Forés s/n, 47003, Valladolid, Spain

    Isabel M. Manjarrés, Pablo Chamero, María Teresa Alonso & Javier García-Sancho

  2. Centro Regional de Investigaciones Biomédicas (CRIB) y Facultad de Medicina, Universidad de Castilla-La Mancha, c/Almansa 14, 02006, Albacete, Spain

    Beatriz Domingo, Francisca Molina & Juan Llopis

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  1. Isabel M. Manjarrés
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  2. Pablo Chamero
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  3. Beatriz Domingo
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  4. Francisca Molina
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  5. Juan Llopis
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  6. María Teresa Alonso
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  7. Javier García-Sancho
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Correspondence to Javier García-Sancho.

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Manjarrés, I.M., Chamero, P., Domingo, B. et al. Red and green aequorins for simultaneous monitoring of Ca2+ signals from two different organelles. Pflugers Arch - Eur J Physiol 455, 961–970 (2008). https://doi.org/10.1007/s00424-007-0349-5

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  • DOI: https://doi.org/10.1007/s00424-007-0349-5

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