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Genetic analysis of circadian responses to low frequency electromagnetic fields in Drosophila melanogaster.

Fedele G, Edwards MD, Bhutani S, Hares JM, Murbach M, Green EW, Dissel S, Hastings MH, Rosato E, Kyriacou CP - PLoS Genet. (2014)

Bottom Line: Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change.In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect.Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, United Kingdom.

ABSTRACT
The blue-light sensitive photoreceptor cryptochrome (CRY) may act as a magneto-receptor through formation of radical pairs involving a triad of tryptophans. Previous genetic analyses of behavioral responses of Drosophila to electromagnetic fields using conditioning, circadian and geotaxis assays have lent some support to the radical pair model (RPM). Here, we describe a new method that generates consistent and reliable circadian responses to electromagnetic fields that differ substantially from those already reported. We used the Schuderer apparatus to isolate Drosophila from local environmental variables, and observe extremely low frequency (3 to 50 Hz) field-induced changes in two locomotor phenotypes, circadian period and activity levels. These field-induced phenotypes are CRY- and blue-light dependent, and are correlated with enhanced CRY stability. Mutational analysis of the terminal tryptophan of the triad hypothesised to be indispensable to the electron transfer required by the RPM reveals that this residue is not necessary for field responses. We observe that deletion of the CRY C-terminus dramatically attenuates the EMF-induced period changes, whereas the N-terminus underlies the hyperactivity. Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change. Finally, we observe that hCRY2, but not hCRY1, transformants can detect EMFs, suggesting that hCRY2 is blue light-responsive. In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect. Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.

No MeSH data available.


Related in: MedlinePlus

Exposure to 500 nm green light lengthens circadian period under EMF.CS flies kept under 500 nm show period lengthening when exposed to EMF compared to sham flies. See Table S1, post-hoc *p<0.05, ***p<0.001). Mean ± sem.
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pgen-1004804-g008: Exposure to 500 nm green light lengthens circadian period under EMF.CS flies kept under 500 nm show period lengthening when exposed to EMF compared to sham flies. See Table S1, post-hoc *p<0.05, ***p<0.001). Mean ± sem.

Mentions: This contradiction may conceivably be resolved by considering the action spectrum of CRY [16], [37] and the ‘antagonistic effect’ of the magnetic field in response to light [38], [39]. Under this proposal, the alignment of the magnetic field would produce inverse or complementary responses under different wavelengths that are dependent on the initial ratio of singlet-triplet states of the radical. This antagonistic effect of wavelength was observed in experiments on magnetic compass orientation in Drosophila, which under green light (500 nm) showed a 90° shift in their alignment compared to flies tested under violet light (365 nm) [18]. This wavelength-dependent effect was also proposed to explain why in the EMF conditioning experiments of Gegear et al. (2008), flies failed to exhibit a response to EMF under full spectrum light when wavelengths below 420 nm were filtered out [38]. As pointed out by Phillips and co-workers, this failure could be due to a change in the nature of the response rather than an inability of the flies to sense the field. Indeed, the response of naïve flies to EMF under full spectrum and full spectrum >420 nm has opposite directions [20]. However, the wavelengths used in our study (430–470 nm) compared to the previous work (445–495 nm [21] and Helfrich-Forster, pers comm)) would initially not appear to be sufficiently different to engage any such antagonistic effect, so the opposite features of the results of the two studies remains puzzling. In an attempt to solve this conundrum, we exposed flies to 500 nm (+/−20 nm) in the Schuderer apparatus, and were surprised to observe that EMF exposed flies revealed a period lengthening rather than the period-shortening we had observed at 450 nm (EMF/Sham Exposure F (1,141) = 5.12, p<0.05 and pre-exposure/exposure F(1,141) = 8.77, p<0.01, Figure 8). Taken together these results at the different wavelengths favor the RPM and the antagonistic model mentioned above, whereby small changes in wavelengths may result in a different Triplet-Singlet ratio and therefore the S-T interconversions would strongly affect the CRY product yield [38]. This striking result nicely explains why the results of Yoshii et al. (2009) are in the opposite direction to ours.


Genetic analysis of circadian responses to low frequency electromagnetic fields in Drosophila melanogaster.

Fedele G, Edwards MD, Bhutani S, Hares JM, Murbach M, Green EW, Dissel S, Hastings MH, Rosato E, Kyriacou CP - PLoS Genet. (2014)

Exposure to 500 nm green light lengthens circadian period under EMF.CS flies kept under 500 nm show period lengthening when exposed to EMF compared to sham flies. See Table S1, post-hoc *p<0.05, ***p<0.001). Mean ± sem.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256086&req=5

pgen-1004804-g008: Exposure to 500 nm green light lengthens circadian period under EMF.CS flies kept under 500 nm show period lengthening when exposed to EMF compared to sham flies. See Table S1, post-hoc *p<0.05, ***p<0.001). Mean ± sem.
Mentions: This contradiction may conceivably be resolved by considering the action spectrum of CRY [16], [37] and the ‘antagonistic effect’ of the magnetic field in response to light [38], [39]. Under this proposal, the alignment of the magnetic field would produce inverse or complementary responses under different wavelengths that are dependent on the initial ratio of singlet-triplet states of the radical. This antagonistic effect of wavelength was observed in experiments on magnetic compass orientation in Drosophila, which under green light (500 nm) showed a 90° shift in their alignment compared to flies tested under violet light (365 nm) [18]. This wavelength-dependent effect was also proposed to explain why in the EMF conditioning experiments of Gegear et al. (2008), flies failed to exhibit a response to EMF under full spectrum light when wavelengths below 420 nm were filtered out [38]. As pointed out by Phillips and co-workers, this failure could be due to a change in the nature of the response rather than an inability of the flies to sense the field. Indeed, the response of naïve flies to EMF under full spectrum and full spectrum >420 nm has opposite directions [20]. However, the wavelengths used in our study (430–470 nm) compared to the previous work (445–495 nm [21] and Helfrich-Forster, pers comm)) would initially not appear to be sufficiently different to engage any such antagonistic effect, so the opposite features of the results of the two studies remains puzzling. In an attempt to solve this conundrum, we exposed flies to 500 nm (+/−20 nm) in the Schuderer apparatus, and were surprised to observe that EMF exposed flies revealed a period lengthening rather than the period-shortening we had observed at 450 nm (EMF/Sham Exposure F (1,141) = 5.12, p<0.05 and pre-exposure/exposure F(1,141) = 8.77, p<0.01, Figure 8). Taken together these results at the different wavelengths favor the RPM and the antagonistic model mentioned above, whereby small changes in wavelengths may result in a different Triplet-Singlet ratio and therefore the S-T interconversions would strongly affect the CRY product yield [38]. This striking result nicely explains why the results of Yoshii et al. (2009) are in the opposite direction to ours.

Bottom Line: Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change.In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect.Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, United Kingdom.

ABSTRACT
The blue-light sensitive photoreceptor cryptochrome (CRY) may act as a magneto-receptor through formation of radical pairs involving a triad of tryptophans. Previous genetic analyses of behavioral responses of Drosophila to electromagnetic fields using conditioning, circadian and geotaxis assays have lent some support to the radical pair model (RPM). Here, we describe a new method that generates consistent and reliable circadian responses to electromagnetic fields that differ substantially from those already reported. We used the Schuderer apparatus to isolate Drosophila from local environmental variables, and observe extremely low frequency (3 to 50 Hz) field-induced changes in two locomotor phenotypes, circadian period and activity levels. These field-induced phenotypes are CRY- and blue-light dependent, and are correlated with enhanced CRY stability. Mutational analysis of the terminal tryptophan of the triad hypothesised to be indispensable to the electron transfer required by the RPM reveals that this residue is not necessary for field responses. We observe that deletion of the CRY C-terminus dramatically attenuates the EMF-induced period changes, whereas the N-terminus underlies the hyperactivity. Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change. Finally, we observe that hCRY2, but not hCRY1, transformants can detect EMFs, suggesting that hCRY2 is blue light-responsive. In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect. Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.

No MeSH data available.


Related in: MedlinePlus