Measuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity calcium Ruby and its dextran conjugate.
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.
Luccardini
Camilla
C
INSERM, U603, Paris F-75006, France
Yakovlev
Aleksey V
AV
Pasche
Mathias
M
Gaillard
Stéphane
S
Li
Dongdong
D
Rousseau
France
F
Ly
Romain
R
Becherer
Ute
U
Mallet
Jean-Maurice
JM
Feltz
Anne
A
Oheim
Martin
M
D000313:Adrenal Medulla / Q000166:cytology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002417:Cattle; D019439:Chromaffin Cells / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D021621:Imaging, Three-Dimensional; D021962:Membrane Microdomains / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
D003911:Dextrans; C067661:Fluoro-Ruby; D012235:Rhodamines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium