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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-induced hyperactivity in cry variants.(A) tim>cry (B) tim>cryΔ;cry02 (C) cry02 (D) tim>cryCT;cry02 (E) tim>cryW342 F;cry02 N's are the same as in Figure 3. Mean ± sem. (see Table S2, post-hoc *p<0.05, **p<0.01, ***p<0.001).
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pgen-1004804-g005: EMF-induced hyperactivity in cry variants.(A) tim>cry (B) tim>cryΔ;cry02 (C) cry02 (D) tim>cryCT;cry02 (E) tim>cryW342 F;cry02 N's are the same as in Figure 3. Mean ± sem. (see Table S2, post-hoc *p<0.05, **p<0.01, ***p<0.001).

Mentions: Similar results were obtained for timgal4>cry overexpressing flies (pre-exposure x EMF/sham exposure interaction (F(1,79) = 4.021, p<0.05, Figure 5A, Table S2) revealing that EMF-exposed flies showed enhanced hyperactivity compared to sham and pre-exposed flies. More surprisingly, timgal4>cryΔ flies also expressed this hyperactivity under EMF exposure (pre-exposure x EMF/sham Exposure interaction F (1,174) = 11.28, p<0.01, Figure 5B, Table S2) whereas no locomotor differences were detected in cry02 (pre-exposure x EMF/sham exposure interaction, F(1, 52) = 0.04, p = 0.95, Figure 5C,Table S2) nor in UASGFPcryCT;timGAL4;cry02 (pre-exposure x EMF/sham interaction, F(1, 118) = 0.51, p = 0.46, Figure 5D, Table S2). Furthermore flies expressing the cryW342F mutation also exhibited the hyperactivity associated with EMF exposure (F(1,54) = 11.9 p<0.01, Figure 5E, Table S2). We therefore conclude that while robust EMF-induced shortening of circadian period requires the CRY C-terminus, the hyperactivity appears to be determined via the N-terminal photolyase-like domain and is not susceptible to disruption by the Trp-342 mutation, indicating that alternative routes are available for the RPM.


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-induced hyperactivity in cry variants.(A) tim>cry (B) tim>cryΔ;cry02 (C) cry02 (D) tim>cryCT;cry02 (E) tim>cryW342 F;cry02 N's are the same as in Figure 3. Mean ± sem. (see Table S2, post-hoc *p<0.05, **p<0.01, ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004804-g005: EMF-induced hyperactivity in cry variants.(A) tim>cry (B) tim>cryΔ;cry02 (C) cry02 (D) tim>cryCT;cry02 (E) tim>cryW342 F;cry02 N's are the same as in Figure 3. Mean ± sem. (see Table S2, post-hoc *p<0.05, **p<0.01, ***p<0.001).
Mentions: Similar results were obtained for timgal4>cry overexpressing flies (pre-exposure x EMF/sham exposure interaction (F(1,79) = 4.021, p<0.05, Figure 5A, Table S2) revealing that EMF-exposed flies showed enhanced hyperactivity compared to sham and pre-exposed flies. More surprisingly, timgal4>cryΔ flies also expressed this hyperactivity under EMF exposure (pre-exposure x EMF/sham Exposure interaction F (1,174) = 11.28, p<0.01, Figure 5B, Table S2) whereas no locomotor differences were detected in cry02 (pre-exposure x EMF/sham exposure interaction, F(1, 52) = 0.04, p = 0.95, Figure 5C,Table S2) nor in UASGFPcryCT;timGAL4;cry02 (pre-exposure x EMF/sham interaction, F(1, 118) = 0.51, p = 0.46, Figure 5D, Table S2). Furthermore flies expressing the cryW342F mutation also exhibited the hyperactivity associated with EMF exposure (F(1,54) = 11.9 p<0.01, Figure 5E, Table S2). We therefore conclude that while robust EMF-induced shortening of circadian period requires the CRY C-terminus, the hyperactivity appears to be determined via the N-terminal photolyase-like domain and is not susceptible to disruption by the Trp-342 mutation, indicating that alternative routes are available for the RPM.

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