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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

EMF exposure increases CRY stability.Top panel. Western blots for CRY using anti-dCRY in wild-type flies expose to EMF or sham in dim blue LL with cry02 and DD control. HSP is used as loading control. Bottom panel. Quantification based on 3 biological replicates each with 3 technical replicates (repeated measures ANOVA F(2,6) = 113.1, p<0.001, post hoc *** p<0.001). Mean ± sem.
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pgen-1004804-g007: EMF exposure increases CRY stability.Top panel. Western blots for CRY using anti-dCRY in wild-type flies expose to EMF or sham in dim blue LL with cry02 and DD control. HSP is used as loading control. Bottom panel. Quantification based on 3 biological replicates each with 3 technical replicates (repeated measures ANOVA F(2,6) = 113.1, p<0.001, post hoc *** p<0.001). Mean ± sem.

Mentions: Western analysis revealed, that levels of CRY in DD were significantly elevated compared to sham in dim blue light as expected [11], but we also observed that under EMF exposure, CRY was significantly more abundant compared to sham (p<0.001, Figure 7). EMF therefore appears to reduce CRY degradation, which in turn would suggest that CRY signalling is compromised.


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)

EMF exposure increases CRY stability.Top panel. Western blots for CRY using anti-dCRY in wild-type flies expose to EMF or sham in dim blue LL with cry02 and DD control. HSP is used as loading control. Bottom panel. Quantification based on 3 biological replicates each with 3 technical replicates (repeated measures ANOVA F(2,6) = 113.1, p<0.001, post hoc *** p<0.001). Mean ± sem.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004804-g007: EMF exposure increases CRY stability.Top panel. Western blots for CRY using anti-dCRY in wild-type flies expose to EMF or sham in dim blue LL with cry02 and DD control. HSP is used as loading control. Bottom panel. Quantification based on 3 biological replicates each with 3 technical replicates (repeated measures ANOVA F(2,6) = 113.1, p<0.001, post hoc *** p<0.001). Mean ± sem.
Mentions: Western analysis revealed, that levels of CRY in DD were significantly elevated compared to sham in dim blue light as expected [11], but we also observed that under EMF exposure, CRY was significantly more abundant compared to sham (p<0.001, Figure 7). EMF therefore appears to reduce CRY degradation, which in turn would suggest that CRY signalling is compromised.

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