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Pflügers Archiv - European Journal of Physiology

, Volume 455, Issue 5, pp 961–970 | Cite as

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

  • Isabel M. Manjarrés
  • Pablo Chamero
  • Beatriz Domingo
  • Francisca Molina
  • Juan Llopis
  • María Teresa Alonso
  • Javier García-SanchoEmail author
Instruments and Techniques

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.

Keywords

Calcium signalling Microdomains Aequorin Chemiluminescence GFP RFP 

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

Notes

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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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Isabel M. Manjarrés
    • 1
  • Pablo Chamero
    • 1
  • Beatriz Domingo
    • 2
  • Francisca Molina
    • 2
  • Juan Llopis
    • 2
  • María Teresa Alonso
    • 1
  • Javier García-Sancho
    • 1
    Email author
  1. 1.Instituto de Biología y Genética Molecular (IBGM)Universidad de Valladolid and Consejo Superior de Investigaciones CientíficasValladolidSpain
  2. 2.Centro Regional de Investigaciones Biomédicas (CRIB) y Facultad de MedicinaUniversidad de Castilla-La ManchaAlbaceteSpain

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