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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

Imaging spontaneous Ca2+ oscillations in Redquorin-expressing zebrafish. a Steps of a typical experiment include mRNA microinjection, reconstitution with CLZ-f, and imaging (hours post-fertilization (hpf)). b Transmitted light and red fluorescence images of embryos expressing Redquorin during early development. c Bioluminescence time course showing spontaneous Ca2+ signals (10 s/frame, whole body field). Selected bioluminescence (relative units ×1,000) and transmitted images are shown. d A single Ca2+ transient in the trunk in the SP2 period, imaged with fast acquisition (integration time was 0.05 s/frame). Average counts in regions of interest on the body and outside of the specimen (background) are shown. Scale bars, 250 μm
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Fig6: Imaging spontaneous Ca2+ oscillations in Redquorin-expressing zebrafish. a Steps of a typical experiment include mRNA microinjection, reconstitution with CLZ-f, and imaging (hours post-fertilization (hpf)). b Transmitted light and red fluorescence images of embryos expressing Redquorin during early development. c Bioluminescence time course showing spontaneous Ca2+ signals (10 s/frame, whole body field). Selected bioluminescence (relative units ×1,000) and transmitted images are shown. d A single Ca2+ transient in the trunk in the SP2 period, imaged with fast acquisition (integration time was 0.05 s/frame). Average counts in regions of interest on the body and outside of the specimen (background) are shown. Scale bars, 250 μm

Mentions: Redquorin from vector pTriEx4-Redquorin was subcloned into EcoRI/XhoI sites of pCS2+ vector for in vitro mRNA synthesis with mMessage mMachine SP6 kit (Ambion). Redquorin mRNA was microinjected into fertilized one-cell stage zebrafish eggs maintained at 28 °C. Injected embryos were dechorionated using pronase (0.5 mg/mL) and forceps. Fluorescence images in Fig. 6b were acquired with an AxioCam MRc color camera (Zeiss) in a Leica MZ16F stereomicroscope using a red filter cube (BP546/12; 560 beamsplitter; BP605/75). Embryos were anesthetized (Tricaine 0.016 %) for prim-16 and long-pec stage images. For standard bioluminescence experiments, dechorionated embryos from 2 to 3 h post-fertilization (hpf) were incubated with 50 μM of CLZ-f or CLZ-hcp (Biotium) in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, and 0.16 mM MgSO4). For bioluminescence imaging, embryos were immobilized in a 0.3 % agarose chamber on a glass-bottom dish and CLZ (20 μM) was maintained during recording. Time-lapse images were acquired in a light-tight microscope (LV200, Olympus), with a ×20 oil-immersion objective (N.A. 0.85), and an EM-CCD camera (CascadeII:512, Photometrics) controlled by MetaMorph software (Molecular Devices). One brightfield image was acquired every 60 bioluminescence images. Recording of the tail contractions shown in Fig. 6d was done in an embryo with the head embeded in 0.3 % agar and a free tail, submerged in E3 medium. Camera was set to slow readout mode or to stream mode (20 fps; 10 MHz readout) for imaging contractions (Fig. 6d). Image analysis was performed with ImageJ (NIH).


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)

Imaging spontaneous Ca2+ oscillations in Redquorin-expressing zebrafish. a Steps of a typical experiment include mRNA microinjection, reconstitution with CLZ-f, and imaging (hours post-fertilization (hpf)). b Transmitted light and red fluorescence images of embryos expressing Redquorin during early development. c Bioluminescence time course showing spontaneous Ca2+ signals (10 s/frame, whole body field). Selected bioluminescence (relative units ×1,000) and transmitted images are shown. d A single Ca2+ transient in the trunk in the SP2 period, imaged with fast acquisition (integration time was 0.05 s/frame). Average counts in regions of interest on the body and outside of the specimen (background) are shown. Scale bars, 250 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig6: Imaging spontaneous Ca2+ oscillations in Redquorin-expressing zebrafish. a Steps of a typical experiment include mRNA microinjection, reconstitution with CLZ-f, and imaging (hours post-fertilization (hpf)). b Transmitted light and red fluorescence images of embryos expressing Redquorin during early development. c Bioluminescence time course showing spontaneous Ca2+ signals (10 s/frame, whole body field). Selected bioluminescence (relative units ×1,000) and transmitted images are shown. d A single Ca2+ transient in the trunk in the SP2 period, imaged with fast acquisition (integration time was 0.05 s/frame). Average counts in regions of interest on the body and outside of the specimen (background) are shown. Scale bars, 250 μm
Mentions: Redquorin from vector pTriEx4-Redquorin was subcloned into EcoRI/XhoI sites of pCS2+ vector for in vitro mRNA synthesis with mMessage mMachine SP6 kit (Ambion). Redquorin mRNA was microinjected into fertilized one-cell stage zebrafish eggs maintained at 28 °C. Injected embryos were dechorionated using pronase (0.5 mg/mL) and forceps. Fluorescence images in Fig. 6b were acquired with an AxioCam MRc color camera (Zeiss) in a Leica MZ16F stereomicroscope using a red filter cube (BP546/12; 560 beamsplitter; BP605/75). Embryos were anesthetized (Tricaine 0.016 %) for prim-16 and long-pec stage images. For standard bioluminescence experiments, dechorionated embryos from 2 to 3 h post-fertilization (hpf) were incubated with 50 μM of CLZ-f or CLZ-hcp (Biotium) in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, and 0.16 mM MgSO4). For bioluminescence imaging, embryos were immobilized in a 0.3 % agarose chamber on a glass-bottom dish and CLZ (20 μM) was maintained during recording. Time-lapse images were acquired in a light-tight microscope (LV200, Olympus), with a ×20 oil-immersion objective (N.A. 0.85), and an EM-CCD camera (CascadeII:512, Photometrics) controlled by MetaMorph software (Molecular Devices). One brightfield image was acquired every 60 bioluminescence images. Recording of the tail contractions shown in Fig. 6d was done in an embryo with the head embeded in 0.3 % agar and a free tail, submerged in E3 medium. Camera was set to slow readout mode or to stream mode (20 fps; 10 MHz readout) for imaging contractions (Fig. 6d). Image analysis was performed with ImageJ (NIH).

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