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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

cry variants alter normal circadian responses to EMFs.Circadian periods (h) in dim blue LL are shown for EMF and sham-exposed groups. Mean periods ± sem. (A) cry02 flies exposed to EMF show only ageing effects on period (yellow shaded box). Wild-type flies kept in DD (grey shaded box) show similar ageing effects (B) tim>cry % rhythmic/arrhythmic flies during pre-exposure and exposure to EMF or sham. Exposure to EMF dramatically increases the proportion of rhythmic flies (χ2(3) = 12.78, p<0.01). (C) tim>cry period for EMF exposed and sham flies before and during exposure (D) tim>cryW342F;cry02 (E) tim>cryΔ;cry02. (F) tim>GFPcryCT;cry02. (See Table S1, post-hoc *p<0.05, **p<0.01, ***p<0.001).
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pgen-1004804-g003: cry variants alter normal circadian responses to EMFs.Circadian periods (h) in dim blue LL are shown for EMF and sham-exposed groups. Mean periods ± sem. (A) cry02 flies exposed to EMF show only ageing effects on period (yellow shaded box). Wild-type flies kept in DD (grey shaded box) show similar ageing effects (B) tim>cry % rhythmic/arrhythmic flies during pre-exposure and exposure to EMF or sham. Exposure to EMF dramatically increases the proportion of rhythmic flies (χ2(3) = 12.78, p<0.01). (C) tim>cry period for EMF exposed and sham flies before and during exposure (D) tim>cryW342F;cry02 (E) tim>cryΔ;cry02. (F) tim>GFPcryCT;cry02. (See Table S1, post-hoc *p<0.05, **p<0.01, ***p<0.001).

Mentions: We therefore pursued our analyses using a 3 Hz/300 µT EMF to study any effect of the cry02 mutation [33]. The response to the EMF was abolished in cry02 flies (Figure 2B, 3A, Table S1), consistent with a possible role for CRY in determining this phenotype (pre-exposure x EMF/sham exposure interaction F(1,52) = 2.93, p = 0.09). However, as mentioned earlier, CRY is required in order to generate the initial blue light-dependent lengthening of period and so these results are not informative in determining whether CRY is the magnetoreceptor. cry02 flies did show a slight lengthening of period between the pre- and exposure conditions of about 0.5 h (F(1,52) = 108.4, p<0.001, Table S1) suggesting an ageing effect over the ∼15 day observation [28]. Indeed we observed a similar period lengthening in CS flies exposed to DD for the same number of days during which CRY would not be light-activated (F(1,54) = 14.40, p<0.001, Figure 3A, Table S1). ANOVA revealed no significant three-way interaction when we compared CS in DD to cry02 in LL (genotype x pre-exposure x EMF/sham exposure, F (2, 106) = 0.07, p = 0.79), supporting the view that the slight lengthening of period was due to ageing. This experiment also clearly shows how the period-shortening of CS flies under EMF is light-dependent (Compare Figure 3A in DD with Figure 1C). Consequently the more dramatic lengthening in period of 1–2 h (Figure 1A–E) observed in CS flies in sham conditions under dim blue LL will also include a small ageing component in addition to that generated by constitutive CRY expression (Table S1). The shortening of period in wild-type flies exposed to EMF is therefore observed in spite of a natural tendency of the flies to increase their period over the duration of the experiment due to ageing (Figure 1, Table S1).


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)

cry variants alter normal circadian responses to EMFs.Circadian periods (h) in dim blue LL are shown for EMF and sham-exposed groups. Mean periods ± sem. (A) cry02 flies exposed to EMF show only ageing effects on period (yellow shaded box). Wild-type flies kept in DD (grey shaded box) show similar ageing effects (B) tim>cry % rhythmic/arrhythmic flies during pre-exposure and exposure to EMF or sham. Exposure to EMF dramatically increases the proportion of rhythmic flies (χ2(3) = 12.78, p<0.01). (C) tim>cry period for EMF exposed and sham flies before and during exposure (D) tim>cryW342F;cry02 (E) tim>cryΔ;cry02. (F) tim>GFPcryCT;cry02. (See Table S1, post-hoc *p<0.05, **p<0.01, ***p<0.001).
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Related In: Results  -  Collection

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

pgen-1004804-g003: cry variants alter normal circadian responses to EMFs.Circadian periods (h) in dim blue LL are shown for EMF and sham-exposed groups. Mean periods ± sem. (A) cry02 flies exposed to EMF show only ageing effects on period (yellow shaded box). Wild-type flies kept in DD (grey shaded box) show similar ageing effects (B) tim>cry % rhythmic/arrhythmic flies during pre-exposure and exposure to EMF or sham. Exposure to EMF dramatically increases the proportion of rhythmic flies (χ2(3) = 12.78, p<0.01). (C) tim>cry period for EMF exposed and sham flies before and during exposure (D) tim>cryW342F;cry02 (E) tim>cryΔ;cry02. (F) tim>GFPcryCT;cry02. (See Table S1, post-hoc *p<0.05, **p<0.01, ***p<0.001).
Mentions: We therefore pursued our analyses using a 3 Hz/300 µT EMF to study any effect of the cry02 mutation [33]. The response to the EMF was abolished in cry02 flies (Figure 2B, 3A, Table S1), consistent with a possible role for CRY in determining this phenotype (pre-exposure x EMF/sham exposure interaction F(1,52) = 2.93, p = 0.09). However, as mentioned earlier, CRY is required in order to generate the initial blue light-dependent lengthening of period and so these results are not informative in determining whether CRY is the magnetoreceptor. cry02 flies did show a slight lengthening of period between the pre- and exposure conditions of about 0.5 h (F(1,52) = 108.4, p<0.001, Table S1) suggesting an ageing effect over the ∼15 day observation [28]. Indeed we observed a similar period lengthening in CS flies exposed to DD for the same number of days during which CRY would not be light-activated (F(1,54) = 14.40, p<0.001, Figure 3A, Table S1). ANOVA revealed no significant three-way interaction when we compared CS in DD to cry02 in LL (genotype x pre-exposure x EMF/sham exposure, F (2, 106) = 0.07, p = 0.79), supporting the view that the slight lengthening of period was due to ageing. This experiment also clearly shows how the period-shortening of CS flies under EMF is light-dependent (Compare Figure 3A in DD with Figure 1C). Consequently the more dramatic lengthening in period of 1–2 h (Figure 1A–E) observed in CS flies in sham conditions under dim blue LL will also include a small ageing component in addition to that generated by constitutive CRY expression (Table S1). The shortening of period in wild-type flies exposed to EMF is therefore observed in spite of a natural tendency of the flies to increase their period over the duration of the experiment due to ageing (Figure 1, Table S1).

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