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, 97 (13), 7260-5

Chimeric Green Fluorescent Protein-Aequorin as Bioluminescent Ca2+ Reporters at the Single-Cell Level

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Chimeric Green Fluorescent Protein-Aequorin as Bioluminescent Ca2+ Reporters at the Single-Cell Level

V Baubet et al. Proc Natl Acad Sci U S A.

Abstract

Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca(2+) stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. Calcium-sensitive bioluminescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca(2+) increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals.

Figures

Figure 1
Figure 1
Schematic map of the different constructions. All of the constructs were under the control of the human cytomegalovirus promoter (PCMV). * indicates the position of the Val-163–Ala mutation. In pGA, the coding sequences of GFP and aequorin are separated by five codons. One to five linkers (in brackets) have been added in pGiA where i is the number of linker. Linkers were oriented so as to encode a 9-aa repeat. Complete synaptotagmin I or its transmembrane part (tSyn) were fused in-frame with the G5A. SV40, simian virus 40.
Figure 2
Figure 2
Ca2+ CRET activities on cellular extracts. Emission spectra of aequorin and several GFP-aequorin fusion proteins were calibrated as a percentage of maximum intensity. CRET measurements are expressed as the ratio of green (500 nm) over blue (450 nm) photons.
Figure 3
Figure 3
GFP fluorescence of GFP-apoaequorin proteins in Neuro2A cells transfected with pGm (A), pGA (B), pG2A (C), and pG5A (D). Confocal superposition of GFP fluorescence and immunostaining of synaptotagmin in cells expressing either pSG5A (E) or pStG5A (F).
Figure 4
Figure 4
Ca2+-induced bioluminescence was detected at the single-cell level. Neuro2A cells transfected with pGA (A.1–A.4) or pSG5A (B) were preincubated with 5 μM coelenterazine in a Ca2+-free buffer (A.3). GFP fluorescence made it possible to choose transfected cells. The background recorded before CaCl2 addition (A.2) corresponds to the RLU level at time 0 of the experiment (A.4 and B). Intensities of fluorescence and bioluminescence activity are translated in scaled pseudocolors. Representative pictures of the chosen field are shown after addition of 5 mM CaCl2 and 5 μM A23187 at 13 sec and 159 sec, respectively, after the beginning of the acquisition (A.1 and A.4). Each profile indicates the intensity of light emitted by a single cell. We defined five regions of interest by encircling individual cell soma. With pGA (data not shown) or pSG5A (B) transfection, a high concentration of CaCl2 (100 mM) was added at the end of the experiment (500 sec) to determine whether the bioluminescent protein was still active (C). Control experiments were made with Fluo-3 AM on mock-transfected Neuro2A cells.

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