Limits...
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

Ca2+ calibration curves of recombinant proteins expressed in E. coli and affinity purified. The relationship between [Ca2+] and (L/Lmax) is displayed on a log–log plot. a Reconstitution with CLZ-native. GA is shown for comparison with permission from Dr. Philippe Brûlet. b Reconstitution with CLZ-f. c Ca2+ titration of Redquorin reconstituted with CLZ-native, CLZ-f, and CLZ-hcp. Dashed vertical lines are drawn at pCa 6
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4537489&req=5

Fig3: Ca2+ calibration curves of recombinant proteins expressed in E. coli and affinity purified. The relationship between [Ca2+] and (L/Lmax) is displayed on a log–log plot. a Reconstitution with CLZ-native. GA is shown for comparison with permission from Dr. Philippe Brûlet. b Reconstitution with CLZ-f. c Ca2+ titration of Redquorin reconstituted with CLZ-native, CLZ-f, and CLZ-hcp. Dashed vertical lines are drawn at pCa 6

Mentions: For a detailed characterization of CitA, tdTA, and Redquorin, we examined the relationship between the fractional rate of luminescence (L/Lmax) and Ca2+ concentration by in vitro calibration (Fig. 3). Recombinant proteins were produced in bacteria, affinity-purified, and reconstituted with CLZ-native, CLZ-f, or CLZ-hcp.Fig. 3


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)

Ca2+ calibration curves of recombinant proteins expressed in E. coli and affinity purified. The relationship between [Ca2+] and (L/Lmax) is displayed on a log–log plot. a Reconstitution with CLZ-native. GA is shown for comparison with permission from Dr. Philippe Brûlet. b Reconstitution with CLZ-f. c Ca2+ titration of Redquorin reconstituted with CLZ-native, CLZ-f, and CLZ-hcp. Dashed vertical lines are drawn at pCa 6
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4537489&req=5

Fig3: Ca2+ calibration curves of recombinant proteins expressed in E. coli and affinity purified. The relationship between [Ca2+] and (L/Lmax) is displayed on a log–log plot. a Reconstitution with CLZ-native. GA is shown for comparison with permission from Dr. Philippe Brûlet. b Reconstitution with CLZ-f. c Ca2+ titration of Redquorin reconstituted with CLZ-native, CLZ-f, and CLZ-hcp. Dashed vertical lines are drawn at pCa 6
Mentions: For a detailed characterization of CitA, tdTA, and Redquorin, we examined the relationship between the fractional rate of luminescence (L/Lmax) and Ca2+ concentration by in vitro calibration (Fig. 3). Recombinant proteins were produced in bacteria, affinity-purified, and reconstituted with CLZ-native, CLZ-f, or CLZ-hcp.Fig. 3

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