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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

Spontaneous Ca2+ oscillations in individual HEK-293 cells visualized with hcp-Redquorin. Cells were imaged with controlled humidity at 37 °C and 5 % CO2 on the microscope stage. The inset shows fluorescence (FL) and bioluminescence images (BL) of two cells corresponding to the traces shown. Scale bar, 20 μm. Integration time was 4 s/frame (representative experiment out of three)
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Fig5: Spontaneous Ca2+ oscillations in individual HEK-293 cells visualized with hcp-Redquorin. Cells were imaged with controlled humidity at 37 °C and 5 % CO2 on the microscope stage. The inset shows fluorescence (FL) and bioluminescence images (BL) of two cells corresponding to the traces shown. Scale bar, 20 μm. Integration time was 4 s/frame (representative experiment out of three)

Mentions: The emission spectrum, emission rate, sensitivity toward Ca2+, and reaction kinetics of Aeq change with the CLZ employed for reconstitution of the apoprotein. Thus, to match the Ca2+ levels of cell compartments or microdomains of high [Ca2+], Aeq Ca2+ affinity can be decreased by using CLZ-n. In addition, it can be further reduced by mutations in the Aeq Ca2+ binding sites [12, 17, 18]. On the other hand, some cells are known to have very low cytosolic [Ca2+] at rest, less than 100 nM [25]. To study such systems, indicators with high Ca2+ affinity should be used [11]. When combined with wt Aeq, CLZ-hcp has been shown to provide the highest emission rate (190-fold more quanta/s than CLZ-native at pCa 7) and fastest kinetics (half-rise 2–4 ms and half-decay 150–300 ms) [27], which are good properties for detecting minute changes occurring during spontaneous Ca2+ oscillations. Our titration data (Figs. 3c and 4b and Table 1) suggests that hcp-Redquorin may provide sufficient sensitivity to measure these low Ca2+ signals. In fact, in unstimulated HEK-293 cells, hcp-Redquorin allowed imaging spontaneous Ca2+ oscillations continuously for up to 5 h (Fig. 5). Thus, Redquorin is able to detect a broad range of Ca2+ levels in single mammalian cells, a demanding application because of the limited light output. It reports agonist-induced cytoplasmic Ca2+ oscillations and also functions as an ultrasensitive indicator, even using non-photon counting detectors (an EM-CCD).Fig. 5


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)

Spontaneous Ca2+ oscillations in individual HEK-293 cells visualized with hcp-Redquorin. Cells were imaged with controlled humidity at 37 °C and 5 % CO2 on the microscope stage. The inset shows fluorescence (FL) and bioluminescence images (BL) of two cells corresponding to the traces shown. Scale bar, 20 μm. Integration time was 4 s/frame (representative experiment out of three)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig5: Spontaneous Ca2+ oscillations in individual HEK-293 cells visualized with hcp-Redquorin. Cells were imaged with controlled humidity at 37 °C and 5 % CO2 on the microscope stage. The inset shows fluorescence (FL) and bioluminescence images (BL) of two cells corresponding to the traces shown. Scale bar, 20 μm. Integration time was 4 s/frame (representative experiment out of three)
Mentions: The emission spectrum, emission rate, sensitivity toward Ca2+, and reaction kinetics of Aeq change with the CLZ employed for reconstitution of the apoprotein. Thus, to match the Ca2+ levels of cell compartments or microdomains of high [Ca2+], Aeq Ca2+ affinity can be decreased by using CLZ-n. In addition, it can be further reduced by mutations in the Aeq Ca2+ binding sites [12, 17, 18]. On the other hand, some cells are known to have very low cytosolic [Ca2+] at rest, less than 100 nM [25]. To study such systems, indicators with high Ca2+ affinity should be used [11]. When combined with wt Aeq, CLZ-hcp has been shown to provide the highest emission rate (190-fold more quanta/s than CLZ-native at pCa 7) and fastest kinetics (half-rise 2–4 ms and half-decay 150–300 ms) [27], which are good properties for detecting minute changes occurring during spontaneous Ca2+ oscillations. Our titration data (Figs. 3c and 4b and Table 1) suggests that hcp-Redquorin may provide sufficient sensitivity to measure these low Ca2+ signals. In fact, in unstimulated HEK-293 cells, hcp-Redquorin allowed imaging spontaneous Ca2+ oscillations continuously for up to 5 h (Fig. 5). Thus, Redquorin is able to detect a broad range of Ca2+ levels in single mammalian cells, a demanding application because of the limited light output. It reports agonist-induced cytoplasmic Ca2+ oscillations and also functions as an ultrasensitive indicator, even using non-photon counting detectors (an EM-CCD).Fig. 5

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