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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

tdTomato-Aequorin (tdTA) variants with modified linker and deletions in tdTomato or Aeq. A scheme of the starting chimera tdTA is shown on top (amino acids in one-letter code). The modifications affected the C-t of tdTomato, the linker, and the N-t of Aeq. The underlined dipeptide linker SG was conserved in most variants and contained insertions in variants L1 and L2 (L stands for long and S for short variants). Horizontal lines above tdTA sequence indicate mutagenic sites for generating different variants. The rightmost column shows the percentage of light of each variant into the 595- and 640-nm emission channels (O + R, orange plus red), measured as indicated in the Materials and methods section. Redquorin-4 and Redquorin-5 contain the Aeq point mutation Y82F (not shown on the sequence)
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Fig1: tdTomato-Aequorin (tdTA) variants with modified linker and deletions in tdTomato or Aeq. A scheme of the starting chimera tdTA is shown on top (amino acids in one-letter code). The modifications affected the C-t of tdTomato, the linker, and the N-t of Aeq. The underlined dipeptide linker SG was conserved in most variants and contained insertions in variants L1 and L2 (L stands for long and S for short variants). Horizontal lines above tdTA sequence indicate mutagenic sites for generating different variants. The rightmost column shows the percentage of light of each variant into the 595- and 640-nm emission channels (O + R, orange plus red), measured as indicated in the Materials and methods section. Redquorin-4 and Redquorin-5 contain the Aeq point mutation Y82F (not shown on the sequence)

Mentions: Variants with different linkers between tdTomato and Aeq were constructed based on tdTA in pCDNA3 (Invitrogen), developed in our previous work [1]. tdTA comprised tdTomato, a 20-amino acid linker and Aeq (Fig. 1). The short linker variants of tdTA (S1, S2, S3, S4, S5, and Redquorin) were obtained by site-directed mutagenesis, converting the sequence coding for some amino acid pairs (marked by a line above tdTA in Fig. 1) into a Kpn2I site, followed by digestion and religation to yield the different clones. The mutagenic oligonucleotides G-D, F224L, and Y82F (Supplementary Table 2) were applied on Redquorin to generate variants Redquorin-2, Redquorin-3, and Redquorin-4, respectively. To construct the long linker variants, a unique Kpn2I restriction site coding for dipeptide SG (underlined in Fig. 1) was placed between the linker and Aeq in tdTA. Complementary oligonucleotides 22aa-L, 13aa-L, and 6aa-L (Supplementary Table 2) were annealed; the resulting double-stranded DNA with overhang extensions were inserted into the Kpn2I site of tdTA and Redquorin-4 to yield variants L1, L2, and Redquorin-5, respectively. The bacterial expression vectors coding for CitA, tdTA, and Redquorin were constructed by digestion of pcDNA3 clones with HindIII and XhoI and subcloning into pTriEX-4 (Novagen) [1]. Restriction enzymes and T4 DNA ligase were from Fermentas, and multi-site mutagenesis kit was from Stratagene. All clones were verified by DNA sequencing.Fig. 1


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)

tdTomato-Aequorin (tdTA) variants with modified linker and deletions in tdTomato or Aeq. A scheme of the starting chimera tdTA is shown on top (amino acids in one-letter code). The modifications affected the C-t of tdTomato, the linker, and the N-t of Aeq. The underlined dipeptide linker SG was conserved in most variants and contained insertions in variants L1 and L2 (L stands for long and S for short variants). Horizontal lines above tdTA sequence indicate mutagenic sites for generating different variants. The rightmost column shows the percentage of light of each variant into the 595- and 640-nm emission channels (O + R, orange plus red), measured as indicated in the Materials and methods section. Redquorin-4 and Redquorin-5 contain the Aeq point mutation Y82F (not shown on the sequence)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: tdTomato-Aequorin (tdTA) variants with modified linker and deletions in tdTomato or Aeq. A scheme of the starting chimera tdTA is shown on top (amino acids in one-letter code). The modifications affected the C-t of tdTomato, the linker, and the N-t of Aeq. The underlined dipeptide linker SG was conserved in most variants and contained insertions in variants L1 and L2 (L stands for long and S for short variants). Horizontal lines above tdTA sequence indicate mutagenic sites for generating different variants. The rightmost column shows the percentage of light of each variant into the 595- and 640-nm emission channels (O + R, orange plus red), measured as indicated in the Materials and methods section. Redquorin-4 and Redquorin-5 contain the Aeq point mutation Y82F (not shown on the sequence)
Mentions: Variants with different linkers between tdTomato and Aeq were constructed based on tdTA in pCDNA3 (Invitrogen), developed in our previous work [1]. tdTA comprised tdTomato, a 20-amino acid linker and Aeq (Fig. 1). The short linker variants of tdTA (S1, S2, S3, S4, S5, and Redquorin) were obtained by site-directed mutagenesis, converting the sequence coding for some amino acid pairs (marked by a line above tdTA in Fig. 1) into a Kpn2I site, followed by digestion and religation to yield the different clones. The mutagenic oligonucleotides G-D, F224L, and Y82F (Supplementary Table 2) were applied on Redquorin to generate variants Redquorin-2, Redquorin-3, and Redquorin-4, respectively. To construct the long linker variants, a unique Kpn2I restriction site coding for dipeptide SG (underlined in Fig. 1) was placed between the linker and Aeq in tdTA. Complementary oligonucleotides 22aa-L, 13aa-L, and 6aa-L (Supplementary Table 2) were annealed; the resulting double-stranded DNA with overhang extensions were inserted into the Kpn2I site of tdTA and Redquorin-4 to yield variants L1, L2, and Redquorin-5, respectively. The bacterial expression vectors coding for CitA, tdTA, and Redquorin were constructed by digestion of pcDNA3 clones with HindIII and XhoI and subcloning into pTriEX-4 (Novagen) [1]. Restriction enzymes and T4 DNA ligase were from Fermentas, and multi-site mutagenesis kit was from Stratagene. All clones were verified by DNA sequencing.Fig. 1

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