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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

hCRY2 but not hCRY1 reveals a sensitivity to EMFs.(A) tim>hCRY1; cry02 or (B) tim>hCRY2; cry02 transformants do not show period shortening under EMF (pre-exposure*EMF/sham interaction hCRY1 F(1,48) = 1.41, p = 0.3 hCRY2 F(1,54) = 0.2, p = 0.63 (see Table S1). (C) hCRY1/2 flies do not show period increase in dim blue LL compared to DD (F(1, 82) = 0.125, p = 0.72) (D) hCRY1 are not hyperactive under EMF (F(1,48) = 0.33, p = 0.56). (E) hCRY2 are hyperactive under EMF exposure. Mean ± sem (see Table S2, post hoc * = p<0.05, ** = p<0.01).
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pgen-1004804-g006: hCRY2 but not hCRY1 reveals a sensitivity to EMFs.(A) tim>hCRY1; cry02 or (B) tim>hCRY2; cry02 transformants do not show period shortening under EMF (pre-exposure*EMF/sham interaction hCRY1 F(1,48) = 1.41, p = 0.3 hCRY2 F(1,54) = 0.2, p = 0.63 (see Table S1). (C) hCRY1/2 flies do not show period increase in dim blue LL compared to DD (F(1, 82) = 0.125, p = 0.72) (D) hCRY1 are not hyperactive under EMF (F(1,48) = 0.33, p = 0.56). (E) hCRY2 are hyperactive under EMF exposure. Mean ± sem (see Table S2, post hoc * = p<0.05, ** = p<0.01).

Mentions: Flies expressing vertebrate non-photoreceptor hCRY2 are reported to exhibit light-dependent magnetoreception in a conditioning assay [25]. By separately expressing tim-GAL4>hCRY1 or hCRY2 on a cry02 background, we observed no significant differences in period between exposed and sham flies (Figure 6A, B, Table S1). Indeed, the hCRY1/2 flies behaved as if they did not respond to dim blue LL because their circadian period does not lengthen in LL compared to DD (Figure 6C), although hCRY proteins have been shown to be light degraded in flies [16] (Fig. S3) and hCRY2 has been implicated in mediating EMF response in a light dependent manner [25]. Nevertheless and somewhat surprisingly, flies expressing hCRY2 but not hCRY1 showed the EMF-induced hyperactivity phenotype (hCRY2 pre-exposure x sham interaction F(1,54) = 5.69 p<0.05, Figure 6D, E, Table S2).


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)

hCRY2 but not hCRY1 reveals a sensitivity to EMFs.(A) tim>hCRY1; cry02 or (B) tim>hCRY2; cry02 transformants do not show period shortening under EMF (pre-exposure*EMF/sham interaction hCRY1 F(1,48) = 1.41, p = 0.3 hCRY2 F(1,54) = 0.2, p = 0.63 (see Table S1). (C) hCRY1/2 flies do not show period increase in dim blue LL compared to DD (F(1, 82) = 0.125, p = 0.72) (D) hCRY1 are not hyperactive under EMF (F(1,48) = 0.33, p = 0.56). (E) hCRY2 are hyperactive under EMF exposure. Mean ± sem (see Table S2, post hoc * = p<0.05, ** = p<0.01).
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pgen-1004804-g006: hCRY2 but not hCRY1 reveals a sensitivity to EMFs.(A) tim>hCRY1; cry02 or (B) tim>hCRY2; cry02 transformants do not show period shortening under EMF (pre-exposure*EMF/sham interaction hCRY1 F(1,48) = 1.41, p = 0.3 hCRY2 F(1,54) = 0.2, p = 0.63 (see Table S1). (C) hCRY1/2 flies do not show period increase in dim blue LL compared to DD (F(1, 82) = 0.125, p = 0.72) (D) hCRY1 are not hyperactive under EMF (F(1,48) = 0.33, p = 0.56). (E) hCRY2 are hyperactive under EMF exposure. Mean ± sem (see Table S2, post hoc * = p<0.05, ** = p<0.01).
Mentions: Flies expressing vertebrate non-photoreceptor hCRY2 are reported to exhibit light-dependent magnetoreception in a conditioning assay [25]. By separately expressing tim-GAL4>hCRY1 or hCRY2 on a cry02 background, we observed no significant differences in period between exposed and sham flies (Figure 6A, B, Table S1). Indeed, the hCRY1/2 flies behaved as if they did not respond to dim blue LL because their circadian period does not lengthen in LL compared to DD (Figure 6C), although hCRY proteins have been shown to be light degraded in flies [16] (Fig. S3) and hCRY2 has been implicated in mediating EMF response in a light dependent manner [25]. Nevertheless and somewhat surprisingly, flies expressing hCRY2 but not hCRY1 showed the EMF-induced hyperactivity phenotype (hCRY2 pre-exposure x sham interaction F(1,54) = 5.69 p<0.05, Figure 6D, E, Table S2).

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