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Light generation of intracellular Ca(2+) signals by a genetically encoded protein BACCS.

Ishii T, Sato K, Kakumoto T, Miura S, Touhara K, Takeuchi S, Nakata T - Nat Commun (2015)

Bottom Line: In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses.Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca(2+).Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca(2+) signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Cell Biology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan [2] The Center for Brain Integration Research, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

ABSTRACT
Ca(2+) signals are highly regulated in a spatiotemporal manner in numerous cellular physiological events. Here we report a genetically engineered blue light-activated Ca(2+) channel switch (BACCS), as an optogenetic tool for generating Ca(2+) signals. BACCS opens Ca(2+)-selective ORAI ion channels in response to light. A BACCS variant, dmBACCS2, combined with Drosophila Orai, elevates the Ca(2+) concentration more rapidly, such that Ca(2+) elevation in mammalian cells is observed within 1 s on light exposure. Using BACCSs, we successfully control cellular events including NFAT-mediated gene expression. In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses. Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca(2+). Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca(2+) signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies.

No MeSH data available.


Related in: MedlinePlus

EOG analyses of odorant- and light-induced responses.(a) Representative fluorescence image of part of the C57BL/6 wild-type (WT) mouse olfactory epithelium infected with the adenovirus (AV) hBACCS2-IRES-mem::tdTomato. Scale bar, 200 μm. (b) Representative fluorescence image of part of the olfactory epithelium from an M71-IRES-ORAI1::hBACCS2-IRES-mem::tdTomato transgenic (TG) mouse. The inset shows a higher-magnification view of the olfactory epithelium. Scale bar, 200 μm. The mice in a and b were 8 weeks of age. (c,d) Light-induced depolarizing potentials recorded from BACCS-expressing olfactory sensory neurons. (c) Average EOG responses of WT mice (left), AV-infected mice (middle) and TG mice (right) to blue light illumination (blue bars). The arrows indicate the timing of the 100-ms amyl acetate application to the olfactory epithelium. Shaded regions around the EOG response traces represent the s.e.m. (d) Box plot of the maximal EOG responses to 4 min of blue light illumination. The y axis represents the EOG responses induced by blue light illumination relative to those induced by amyl acetate exposures. The data shown are for n=10 WT mice, n=16 AV-infected mice and n=12 TG mice. Significance was assessed by t-test: *P<0.05, **P<0.01.
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f6: EOG analyses of odorant- and light-induced responses.(a) Representative fluorescence image of part of the C57BL/6 wild-type (WT) mouse olfactory epithelium infected with the adenovirus (AV) hBACCS2-IRES-mem::tdTomato. Scale bar, 200 μm. (b) Representative fluorescence image of part of the olfactory epithelium from an M71-IRES-ORAI1::hBACCS2-IRES-mem::tdTomato transgenic (TG) mouse. The inset shows a higher-magnification view of the olfactory epithelium. Scale bar, 200 μm. The mice in a and b were 8 weeks of age. (c,d) Light-induced depolarizing potentials recorded from BACCS-expressing olfactory sensory neurons. (c) Average EOG responses of WT mice (left), AV-infected mice (middle) and TG mice (right) to blue light illumination (blue bars). The arrows indicate the timing of the 100-ms amyl acetate application to the olfactory epithelium. Shaded regions around the EOG response traces represent the s.e.m. (d) Box plot of the maximal EOG responses to 4 min of blue light illumination. The y axis represents the EOG responses induced by blue light illumination relative to those induced by amyl acetate exposures. The data shown are for n=10 WT mice, n=16 AV-infected mice and n=12 TG mice. Significance was assessed by t-test: *P<0.05, **P<0.01.

Mentions: To examine whether BACCS is applicable to mouse tissues, we generated a recombinant adenovirus for expression of hBACCS2-IRES-mem::tdTomato, and infected the adenovirus into the olfactory epithelium (Fig. 6a). We recorded local field potentials from the surface of the olfactory epithelium in an ex vivo whole-mount preparation, the electro-olfactogram (EOG)43. Figure 6c shows the waveforms of the average EOG responses to blue light and the odorant amyl acetate as a positive control. Significant responses to both blue light and amyl acetate were recorded at the infected surface of the olfactory epithelium on blue light exposure, while the olfactory epithelium of wild-type mice only showed responses to amyl acetate (Fig. 6c,d), suggesting that hBACCS2 was photoactivated in olfactory sensory neurons (OSNs).


Light generation of intracellular Ca(2+) signals by a genetically encoded protein BACCS.

Ishii T, Sato K, Kakumoto T, Miura S, Touhara K, Takeuchi S, Nakata T - Nat Commun (2015)

EOG analyses of odorant- and light-induced responses.(a) Representative fluorescence image of part of the C57BL/6 wild-type (WT) mouse olfactory epithelium infected with the adenovirus (AV) hBACCS2-IRES-mem::tdTomato. Scale bar, 200 μm. (b) Representative fluorescence image of part of the olfactory epithelium from an M71-IRES-ORAI1::hBACCS2-IRES-mem::tdTomato transgenic (TG) mouse. The inset shows a higher-magnification view of the olfactory epithelium. Scale bar, 200 μm. The mice in a and b were 8 weeks of age. (c,d) Light-induced depolarizing potentials recorded from BACCS-expressing olfactory sensory neurons. (c) Average EOG responses of WT mice (left), AV-infected mice (middle) and TG mice (right) to blue light illumination (blue bars). The arrows indicate the timing of the 100-ms amyl acetate application to the olfactory epithelium. Shaded regions around the EOG response traces represent the s.e.m. (d) Box plot of the maximal EOG responses to 4 min of blue light illumination. The y axis represents the EOG responses induced by blue light illumination relative to those induced by amyl acetate exposures. The data shown are for n=10 WT mice, n=16 AV-infected mice and n=12 TG mice. Significance was assessed by t-test: *P<0.05, **P<0.01.
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Related In: Results  -  Collection

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f6: EOG analyses of odorant- and light-induced responses.(a) Representative fluorescence image of part of the C57BL/6 wild-type (WT) mouse olfactory epithelium infected with the adenovirus (AV) hBACCS2-IRES-mem::tdTomato. Scale bar, 200 μm. (b) Representative fluorescence image of part of the olfactory epithelium from an M71-IRES-ORAI1::hBACCS2-IRES-mem::tdTomato transgenic (TG) mouse. The inset shows a higher-magnification view of the olfactory epithelium. Scale bar, 200 μm. The mice in a and b were 8 weeks of age. (c,d) Light-induced depolarizing potentials recorded from BACCS-expressing olfactory sensory neurons. (c) Average EOG responses of WT mice (left), AV-infected mice (middle) and TG mice (right) to blue light illumination (blue bars). The arrows indicate the timing of the 100-ms amyl acetate application to the olfactory epithelium. Shaded regions around the EOG response traces represent the s.e.m. (d) Box plot of the maximal EOG responses to 4 min of blue light illumination. The y axis represents the EOG responses induced by blue light illumination relative to those induced by amyl acetate exposures. The data shown are for n=10 WT mice, n=16 AV-infected mice and n=12 TG mice. Significance was assessed by t-test: *P<0.05, **P<0.01.
Mentions: To examine whether BACCS is applicable to mouse tissues, we generated a recombinant adenovirus for expression of hBACCS2-IRES-mem::tdTomato, and infected the adenovirus into the olfactory epithelium (Fig. 6a). We recorded local field potentials from the surface of the olfactory epithelium in an ex vivo whole-mount preparation, the electro-olfactogram (EOG)43. Figure 6c shows the waveforms of the average EOG responses to blue light and the odorant amyl acetate as a positive control. Significant responses to both blue light and amyl acetate were recorded at the infected surface of the olfactory epithelium on blue light exposure, while the olfactory epithelium of wild-type mice only showed responses to amyl acetate (Fig. 6c,d), suggesting that hBACCS2 was photoactivated in olfactory sensory neurons (OSNs).

Bottom Line: In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses.Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca(2+).Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca(2+) signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Cell Biology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan [2] The Center for Brain Integration Research, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

ABSTRACT
Ca(2+) signals are highly regulated in a spatiotemporal manner in numerous cellular physiological events. Here we report a genetically engineered blue light-activated Ca(2+) channel switch (BACCS), as an optogenetic tool for generating Ca(2+) signals. BACCS opens Ca(2+)-selective ORAI ion channels in response to light. A BACCS variant, dmBACCS2, combined with Drosophila Orai, elevates the Ca(2+) concentration more rapidly, such that Ca(2+) elevation in mammalian cells is observed within 1 s on light exposure. Using BACCSs, we successfully control cellular events including NFAT-mediated gene expression. In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses. Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca(2+). Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca(2+) signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies.

No MeSH data available.


Related in: MedlinePlus