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Imaging Ca(2+) activity in mammalian cells and zebrafish with a novel red-emitting aequorin variant.

Bakayan A, Domingo B, Miyawaki A, Llopis J - Pflugers Arch. (2014)

Bottom Line: In addition, we also imaged Ca(2+) transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period.Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue.This new tool will facilitate in vivo imaging of Ca(2+) from deep tissues of animals.

View Article: PubMed Central - PubMed

Affiliation: Centro Regional de Investigaciones Biomédicas (CRIB) and Facultad de Medicina de Albacete, Universidad Castilla-La Mancha, C/ Almansa 14, 02008, Albacete, Spain.

ABSTRACT
Ca(2+) monitoring with aequorin is an established bioluminescence technique, whereby the photoprotein emits blue light when it binds to Ca(2+). However, aequorin's blue emission and low quantum yield limit its application for in vivo imaging because blue-green light is greatly attenuated in animal tissues. In earlier work, aequorin was molecularly fused with green, yellow, and red fluorescent proteins, producing an emission shift through bioluminescence resonance energy transfer (BRET). We have previously shown that the chimera tandem dimer Tomato-aequorin (tdTA) emits red light in mammalian cells and across the skin and other tissues of mice [1]. In this work, we varied the configuration of the linker in tdTA to maximize energy transfer. One variant, named Redquorin, improved BRET from aequorin to tdTomato to almost a maximum value, and the emission above 575 nm exceeded 73 % of total counts. By pairing Redquorin with appropriate synthetic coelenterazines, agonist-induced and spontaneous Ca(2+) oscillations in single HEK-293 cells were imaged. In addition, we also imaged Ca(2+) transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period. Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue. This new tool will facilitate in vivo imaging of Ca(2+) from deep tissues of animals.

No MeSH data available.


Related in: MedlinePlus

In vitro characterization of tdTA variants. a Ca2+-triggered bioluminescence spectra of fluorescent protein (FP)-Aeq chimeras. The hybrid proteins, obtained from HeLa cell lysates (GA) or produced in E. coli and affinity column-purified (CitA, tdTA, Redquorin) were reconstituted with CLZ-f. The sum of the height of the two peaks (Aeq and fluorescent protein) was normalized to one. The inset indicates the wavelength of Aeq (donor) and FP (acceptor) emission peaks and the full width at half-maximum (FWHM) of the FP peak, all expressed in nanometers. b The integrity of affinity-purified hybrid proteins CitA, tdTA, and Redquorin was tested on a native gel and visualized by fluorescence (left), followed by Coomassie staining (right). c Bioluminescence spectra per picomole of f-CitA and f-Redquorin suspended in buffer or blood (blood dilution after addition of Ca2+ was 4.5-fold). d Transmittance spectrum of a oxyhemoglobin solution. e Comparison of f-CitA and f-Redquorin spectra obtained experimentally in blood (dotted lines) with the calculated spectra considering the transmittance of oxyhemoglobin (full lines). The dashed vertical lines are shown to highlight the luminescence above 590 nm
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Fig2: In vitro characterization of tdTA variants. a Ca2+-triggered bioluminescence spectra of fluorescent protein (FP)-Aeq chimeras. The hybrid proteins, obtained from HeLa cell lysates (GA) or produced in E. coli and affinity column-purified (CitA, tdTA, Redquorin) were reconstituted with CLZ-f. The sum of the height of the two peaks (Aeq and fluorescent protein) was normalized to one. The inset indicates the wavelength of Aeq (donor) and FP (acceptor) emission peaks and the full width at half-maximum (FWHM) of the FP peak, all expressed in nanometers. b The integrity of affinity-purified hybrid proteins CitA, tdTA, and Redquorin was tested on a native gel and visualized by fluorescence (left), followed by Coomassie staining (right). c Bioluminescence spectra per picomole of f-CitA and f-Redquorin suspended in buffer or blood (blood dilution after addition of Ca2+ was 4.5-fold). d Transmittance spectrum of a oxyhemoglobin solution. e Comparison of f-CitA and f-Redquorin spectra obtained experimentally in blood (dotted lines) with the calculated spectra considering the transmittance of oxyhemoglobin (full lines). The dashed vertical lines are shown to highlight the luminescence above 590 nm

Mentions: We obtained the Ca2+-dependent luminescence spectrum of affinity-purified Redquorin (Fig. 2a) and earlier FP-Aeq fusions. The integrity of the samples was verified by fluorescence on a native gel (Fig. 2b). The 582-nm peak of Redquorin, due to BRET, was much larger than the corresponding peak of tdTA, and there was an Aeq residual peak at 470 nm. In addition, since the main peak of Redquorin was wider than that of GA or CitA (full width at half-maximum of 70 nm, compared to 38 nm) (Fig. 2a, inset), there was a significant amount of counts above 590 nm, in the optical window for intravital imaging in mammals. Western blots of cytosolic extracts of HeLa cells transfected with tdTA and Redquorin showed unique bands of the expected molecular mass (Supplementary Fig. 2), suggesting that the chimeric proteins were stable within cells.Fig. 2


Imaging Ca(2+) activity in mammalian cells and zebrafish with a novel red-emitting aequorin variant.

Bakayan A, Domingo B, Miyawaki A, Llopis J - Pflugers Arch. (2014)

In vitro characterization of tdTA variants. a Ca2+-triggered bioluminescence spectra of fluorescent protein (FP)-Aeq chimeras. The hybrid proteins, obtained from HeLa cell lysates (GA) or produced in E. coli and affinity column-purified (CitA, tdTA, Redquorin) were reconstituted with CLZ-f. The sum of the height of the two peaks (Aeq and fluorescent protein) was normalized to one. The inset indicates the wavelength of Aeq (donor) and FP (acceptor) emission peaks and the full width at half-maximum (FWHM) of the FP peak, all expressed in nanometers. b The integrity of affinity-purified hybrid proteins CitA, tdTA, and Redquorin was tested on a native gel and visualized by fluorescence (left), followed by Coomassie staining (right). c Bioluminescence spectra per picomole of f-CitA and f-Redquorin suspended in buffer or blood (blood dilution after addition of Ca2+ was 4.5-fold). d Transmittance spectrum of a oxyhemoglobin solution. e Comparison of f-CitA and f-Redquorin spectra obtained experimentally in blood (dotted lines) with the calculated spectra considering the transmittance of oxyhemoglobin (full lines). The dashed vertical lines are shown to highlight the luminescence above 590 nm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4537489&req=5

Fig2: In vitro characterization of tdTA variants. a Ca2+-triggered bioluminescence spectra of fluorescent protein (FP)-Aeq chimeras. The hybrid proteins, obtained from HeLa cell lysates (GA) or produced in E. coli and affinity column-purified (CitA, tdTA, Redquorin) were reconstituted with CLZ-f. The sum of the height of the two peaks (Aeq and fluorescent protein) was normalized to one. The inset indicates the wavelength of Aeq (donor) and FP (acceptor) emission peaks and the full width at half-maximum (FWHM) of the FP peak, all expressed in nanometers. b The integrity of affinity-purified hybrid proteins CitA, tdTA, and Redquorin was tested on a native gel and visualized by fluorescence (left), followed by Coomassie staining (right). c Bioluminescence spectra per picomole of f-CitA and f-Redquorin suspended in buffer or blood (blood dilution after addition of Ca2+ was 4.5-fold). d Transmittance spectrum of a oxyhemoglobin solution. e Comparison of f-CitA and f-Redquorin spectra obtained experimentally in blood (dotted lines) with the calculated spectra considering the transmittance of oxyhemoglobin (full lines). The dashed vertical lines are shown to highlight the luminescence above 590 nm
Mentions: We obtained the Ca2+-dependent luminescence spectrum of affinity-purified Redquorin (Fig. 2a) and earlier FP-Aeq fusions. The integrity of the samples was verified by fluorescence on a native gel (Fig. 2b). The 582-nm peak of Redquorin, due to BRET, was much larger than the corresponding peak of tdTA, and there was an Aeq residual peak at 470 nm. In addition, since the main peak of Redquorin was wider than that of GA or CitA (full width at half-maximum of 70 nm, compared to 38 nm) (Fig. 2a, inset), there was a significant amount of counts above 590 nm, in the optical window for intravital imaging in mammals. Western blots of cytosolic extracts of HeLa cells transfected with tdTA and Redquorin showed unique bands of the expected molecular mass (Supplementary Fig. 2), suggesting that the chimeric proteins were stable within cells.Fig. 2

Bottom Line: In addition, we also imaged Ca(2+) transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period.Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue.This new tool will facilitate in vivo imaging of Ca(2+) from deep tissues of animals.

View Article: PubMed Central - PubMed

Affiliation: Centro Regional de Investigaciones Biomédicas (CRIB) and Facultad de Medicina de Albacete, Universidad Castilla-La Mancha, C/ Almansa 14, 02008, Albacete, Spain.

ABSTRACT
Ca(2+) monitoring with aequorin is an established bioluminescence technique, whereby the photoprotein emits blue light when it binds to Ca(2+). However, aequorin's blue emission and low quantum yield limit its application for in vivo imaging because blue-green light is greatly attenuated in animal tissues. In earlier work, aequorin was molecularly fused with green, yellow, and red fluorescent proteins, producing an emission shift through bioluminescence resonance energy transfer (BRET). We have previously shown that the chimera tandem dimer Tomato-aequorin (tdTA) emits red light in mammalian cells and across the skin and other tissues of mice [1]. In this work, we varied the configuration of the linker in tdTA to maximize energy transfer. One variant, named Redquorin, improved BRET from aequorin to tdTomato to almost a maximum value, and the emission above 575 nm exceeded 73 % of total counts. By pairing Redquorin with appropriate synthetic coelenterazines, agonist-induced and spontaneous Ca(2+) oscillations in single HEK-293 cells were imaged. In addition, we also imaged Ca(2+) transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period. Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue. This new tool will facilitate in vivo imaging of Ca(2+) from deep tissues of animals.

No MeSH data available.


Related in: MedlinePlus