Animal cryptochromes mediate magnetoreception by an unconventional photochemical mechanism

Abstract

Understanding the biophysical basis of animal magnetoreception has been one of the greatest challenges in sensory biology. Recently it was discovered that the light-dependent magnetic sense of Drosophila melanogaster is mediated by the ultraviolet (UV)-A/blue light photoreceptor cryptochrome (Cry). Here we show, using a transgenic approach, that the photoreceptive, Drosophila-like type 1 Cry and the transcriptionally repressive, vertebrate-like type 2 Cry of the monarch butterfly (Danaus plexippus) can both function in the magnetoreception system of Drosophila and require UV-A/blue light (wavelength below 420 nm) to do so. The lack of magnetic responses for both Cry types at wavelengths above 420 nm does not fit the widely held view that tryptophan triad-generated radical pairs mediate the ability of Cry to sense a magnetic field. We bolster this assessment by using a mutant form of Drosophila and monarch type 1 Cry and confirm that the tryptophan triad pathway is not crucial in magnetic transduction. Together, these results suggest that animal Crys mediate light-dependent magnetoreception through an unconventional photochemical mechanism. This work emphasizes the utility of Drosophila transgenesis for elucidating the precise mechanisms of Cry-mediated magnetosensitivity in insects and also in vertebrates such as migrating birds.

Figure 1. Type 1 Crys rescue light-dependent magnetoreception in Cry-deficient flies

a, A tim-GAL4 driven Drosophila transgene (tim-GAL4/UAS-dcry) rescues magnetic responses in cry^b flies, similar to the responses of wild-type Canton-S flies, while tim-GAL4/+ or UAS-dcry/+ alone do not. Bars show preference index values for naïve (white) and trained (black) groups. Numbers represent groups tested. *, P<0.05; ***, P<0.001. Genotypes in parentheses: tim-GAL4/+ (y w; tim-GAL4/+; cry^b), UAS-dcry/+ (y w; UAS-mycdcry/+; cry^b), and tim-GAL4/UAS-dcry (y w; tim-GAL4/UAS-mycdcry; cry^b). b, A tim-GAL4 driven monarch (dp)cry1 transgene (tim-GAL4/ UAS-dpcry1) rescues magnetic responses in cry^b flies, while tim-GAL4/+ or UAS-dpcry1/+ alone do not. Bars show preference index values for naïve (white) and trained (black) groups. **, P<0.01. Genotypes in parentheses: tim-GAL4/+ (y w; tim-GAL4/+; cry^b), UAS-dpcry1/+ (y w; UAS-mycdpCry1#15b/+; cry^b), and tim-GAL4/ UAS-dpcry1. c, Irradiance curves for different light conditions. Light measurements were taken from inside the training and test tube. Full-spectrum and >420 nm irradiance curves were reported before. d, Wavelength-dependence of magnetic responses is rescued by monarch (dp)Cry1 (y w; tim-GAL4/UAS-mycdpCry1#15b; cry^b). The full-spectrum data are the same as those depicted in b. Bars show preference index values for naïve (white) and trained (black) groups. *, P<0.05, **, P<0.01. Values from a, b, and d are mean ± s.e.m.

Figure 2. Monarch Type 2 Cry rescues light-dependent magnetosensivitiy in Cry-deficient flies

a, A tim-GAL4 driven monarch (dp)cry2 transgene (tim-GAL4/ UAS-dpcry2) rescues magnetic responses in cry^b flies, while UAS-dpcry2/+ alone does not. The tim-GAL4/+ data are re-plotted from Fig. 1b. Bars show preference index values for naïve (white) and trained (black) groups. Numbers represent groups tested. *, P<0.05. Genotypes in parentheses: tim-GAL4/+ (y w; tim-GAL4/+; cry^b), UAS-dpcry2/+ (y w; UAS- mycdpCry2#125a/+; cry^b), and tim-GAL4/ UAS-dpcry2 (y w; tim-GAL4/UAS- mycdpCry2#125a; cry^b). b, Wavelength-dependence of magnetic responses is rescued by monarch (dp)Cry2 (y w; tim-GAL4/UAS- mycdpCry2#125a; cry^b). The full spectrum data are the same that those depicted in a. Bars show preference index values for naïve (white) and trained (black) groups. *, P<0.05. Values from a and b are mean ± s.e.m.

Figure 3. Effects of terminal tryptophan mutations on Type 1 and Type 2 Cry-mediated magnetosensitivity

a, The Drosophila CryW342F mutation rescues magnetosensitive responses. Two mutant lines (111a and 132a) of y w; tim-GAL4/UAS-dcryW342F; cry^b were tested. Bars show preference index values for naïve (white) and trained (black) groups. Numbers represent groups tested. *, P<0.05, **, P<0.01. b, The monarch Cry1W328F mutation rescues magnetosensitive responses. Three mutant lines (126a, 109a, and 18a) of y w; tim-GAL4/UAS-dpcry1W328F; cry^b were tested. Bars show preference index values for naïve (white) and trained (black) groups. Numbers represent groups tested. *, P<0.05, **, P<0.01; ***, P<0.001. c, The monarch Cry2W345F mutation does not rescue magnetosensitive responses. Two mutant lines (130a and 131) of y w; tim-GAL4/ UAS-dpcry2W345F; cry^b were tested. Bars show preference index of the naïve (white) and trained (black) groups. Numbers represent groups tested. For a–c, none of the UAS-trp mutant/+ lines (without driver) restored magnetosensitive behavioural responses (data not shown). Values from a–c are mean ± s.e.m. All Cry mutations were sequenced and confirmed from all of the different transgenic lines used. d, Cry2W345F still inhibits Clock:Cycle-mediated transcription in Schneider 2 cells. The dpPerEp reporter (20 ng) was tested in presence (+) or absence (−) of dpClk/dpCyc expression plasmids (10 ng each); wild-type dpCry2 (20, 50 and 100 ng) or dpCry2W345F (20, 50 and 100 ng) were used,. Luciferase activity relative to β-galactosidase activity was computed. Lower, representative western blot of Cry2 (dpCry2 or dpCry2W345F) and tubulin expression. Values are mean ± s.e.m for three independent experiments.

Figure 4. The clock proteins Tim and Cyc are not required for Drosophila magnetoreception

Naïve and trained responses to a magnetic field are not impaired in either Tim-deficient tim⁰ mutant flies or Cyc-deficient cyc⁰ flies. Bars show preference index values for naïve (white) and trained (black) groups. Numbers represent groups tested. Values are mean ± s.e.m. *, P<0.05; **, P<0.01; ****, P<0.0001.