Measuring mitochondrial and cytoplasmic Ca super(2) super(+) in EGFP expressing cells with a low-affinity Calcium Ruby and its dextran conjugate

• Published in: Cell calcium (Edinburgh) 2009-03, Vol.45 (3), p.275-283
• Main Authors: Luccardini, C, Yakovlev, A V, Pasche, M, Gaillard, S, Li, D, Rousseau, F, Ly, R, Becherer, U, Mallet, J M, Feltz, A, Oheim, M
• Format: Article
• Language: English
• ISSN: 0143-4160
• DOI: 10.1016/j.ceca.2008.11.007

The limited choice and poor performance of red-emitting calcium (Ca super(2) super(+)) indicators have hampered microfluorometric measurements of the intracellular free Ca super(2) super(+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca super(2) super(+) 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 (578/602nm peak excitation/emission) conjugated to BAPTA and having an additional NH sub(2) linker arm. The low-affinity variant (K sub(D) sub(,) sub(C) sub(a)~30 mu M) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca super(2) super(+) transients. While Calcium Ruby is a mitochondrial Ca super(2) super(+)probe, its conjugation, via the NH sub(2) tail, to a 10,000MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca super(2) super(+) probe with an affinity matched to microdomain Ca super(2) super(+) signals. As an example, we show depolarization-evoked Ca super(2) super(+) 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 super(2) super(+) measurements.