Measuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity calcium Ruby and its dextran conjugate. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Cell Calcium Année : 2009

Measuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity calcium Ruby and its dextran conjugate.

C. Luccardini,
  • Fonction : Auteur
A. Yakovlev,
  • Fonction : Auteur
M. Pasche,
  • Fonction : Auteur
S. Gaillard,
  • Fonction : Auteur
D. D. Li,
  • Fonction : Auteur
F. Rousseau,
  • Fonction : Auteur
R. Ly,
  • Fonction : Auteur
U. Betcherer,
  • Fonction : Auteur
A. Feltz,
  • Fonction : Auteur
M. Oheim,
  • Fonction : Auteur

Résumé

The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered microfluorometric measurements of the intracellular free Ca(2+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca(2+) indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH(2) linker arm. The low-affinity variant (K(D,Ca) approximately 30 microM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca(2+) transients. While Calcium Ruby is a mitochondrial Ca(2+)probe, its conjugation, via the NH(2) tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca(2+) probe with an affinity matched to microdomain Ca(2+) signals. As an example, we show depolarization-evoked Ca(2+) signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca(2+) measurements.

Domaines

Chimie organique

Dates et versions

hal-00411198 , version 1 (26-08-2009)

Identifiants

Citer

C. Luccardini,, A. Yakovlev,, M. Pasche,, S. Gaillard,, D. D. Li,, et al.. Measuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity calcium Ruby and its dextran conjugate.. Cell Calcium, 2009, 45 (3), pp.275-83. ⟨10.1016/j.ceca.2008.11.007⟩. ⟨hal-00411198⟩
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