Cytotechnology

, Volume 11, Supplement 1, pp S44–S46

Intracellular targeting of the photoprotein aequorin: A new approach for measuring, in living cells, Ca2+ concentrations in defined cellular compartments

  • Rosario Rizzuto
  • Marisa Brini
  • Tullio Pozzan
Session 2 Intracellular Architecture and Traffic Invited Lectures

DOI: 10.1007/BF00746051

Cite this article as:
Rizzuto, R., Brini, M. & Pozzan, T. Cytotechnology (1993) 11(Suppl 1): S44. doi:10.1007/BF00746051

Abstract

We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca2+ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a “mitochondrially-targeted” aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca2+, [Ca2+]m, were obtained, which showed that [Ca2+]m is low at rest (<0.5 μM), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria “sense” very accurately the cytosolic Ca2+ concentration ([Ca2+]i), and after cell stimulation [Ca2+]m rises to values capable of activating the Ca2+-sensitive mitochondrial dehydrogenases.

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Rosario Rizzuto
    • 1
  • Marisa Brini
    • 1
  • Tullio Pozzan
    • 1
  1. 1.Dept. Biomedical Sciences and CNR Center for the Study of Mitochondrial PhysiologyUniversity of PadovaPadovaItaly