Abstract
Night-migratory songbirds are remarkably proficient navigators1. Flying alone and often over great distances, they use various directional cues including, crucially, a light-dependent magnetic compass2,3. The mechanism of this compass has been suggested to rely on the quantum spin dynamics of photoinduced radical pairs in cryptochrome flavoproteins located in the retinas of the birds4,5,6,7. Here we show that the photochemistry of cryptochrome 4 (CRY4) from the night-migratory European robin (Erithacus rubecula) is magnetically sensitive in vitro, and more so than CRY4 from two non-migratory bird species, chicken (Gallus gallus) and pigeon (Columba livia). Site-specific mutations of ErCRY4 reveal the roles of four successive flavin–tryptophan radical pairs in generating magnetic field effects and in stabilizing potential signalling states in a way that could enable sensing and signalling functions to be independently optimized in night-migratory birds.
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Data availability
The complete set of molecular dynamics simulation and quantum chemistry data (300 GB) can be downloaded from the University of Oldenburg repository: https://cloud.uol.de/s/NrTYpoEzL6RbPq7. Specific molecular dynamics data can also be obtained directly from I.A.S. on request. Source data are provided with this paper.
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Acknowledgements
This work was supported by the Air Force Office of Scientific Research (Air Force Materiel Command, USAF award no. FA9550-14-1-0095, to P.J.H., H.M., C.R.T., S.R.M. and K.-W.K.); by the European Research Council (under the European Union’s Horizon 2020 research and innovation programme, grant agreement no. 810002, Synergy Grant: ‘QuantumBirds’, awarded to P.J.H. and H.M.); by the Office of Naval Research Global, award no. N62909-19-1-2045, to P.J.H., C.R.T. and S.R.M.; by the Deutsche Forschungsgemeinschaft (SFB 1372, ‘Magnetoreception and navigation in vertebrates’, project number: 395940726 to H.M., K.-W.K., I.A.S. and P.J.H., and GRK 1885, ‘Molecular basis of sensory biology’ to K.-W.K., I.A.S. and H.M.); by a DAAD (German Academic Exchange Service, Graduate School Scholarship Programme, ID 57395813) stipend to J.X.; by funding for G.M. from the SCG Innovation Fund; by the Electromagnetic Fields Biological Research Trust (to P.J.H., C.R.T. and S.R.M.); by the National Natural Science Foundation of China, grant no. 31640001, and the Presidential Foundation of Hefei Institutes of Physical Science, Chinese Academy of Sciences, grant no. BJZX201901 (to C.X.); and by the Lundbeck Foundation, the Danish Councils for Independent Research, and the Volkswagen Foundation (to I.A.S.). V.D. is grateful to the Clarendon Fund, University of Oxford. M.J.G. thanks the Biotechnology and Biological Sciences Research Council, grant number BB/M011224/1 and the Clarendon Fund. We acknowledge use of the Advanced Research Computing (ARC) facility of the University of Oxford. J.S.T. is an Investigator and Y.C. is a Research Specialist in the Howard Hughes Medical Institute. I.A.S. is grateful to the DeiC National HPC Center, University of Southern Denmark for computational resources. We thank B. Grünberg, I. Fomins, A. Günther and A. Einwich for laboratory assistance and for providing protein sequence information. J.X. thanks Y. Tan for training in protein expression and purification. We thank S. Chandler for mass spectrometry, S. Y. Wong for assistance with spin dynamics calculations, and W. Myers (CAESR, Engineering and Physical Sciences Research Council, grant no. EP/L011972/1) for obtaining a threefold improvement in the time-resolved electron paramagnetic resonance signal. We are grateful to the staff of the mechanical and electronic workshops in the Oxford Chemistry Department and at the University of Oldenburg.
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J.X., L.E.J., T.Z., M.K., K.B.H., S.R. and M.J.G. made particularly important experimental contributions. J.X. cloned wild-type ErCRY4 and all the mutants and developed the protocols for expression and purification of the proteins with FAD bound. J.X. and J.S. produced the protein samples. L.E.J. developed the continuous illumination experiment for studying photoreduction and the picosecond transient absorption experiment for measuring magnetic field effects, and recorded and interpreted data. T.Z. and M.J.G. developed the CRDS experiment for measuring magnetic field effects and recorded and interpreted data. M.K. developed the broadband cavity-enhanced absorption spectroscopy experiment for measuring magnetic field effects and recorded and interpreted data. S.R. and S.W. recorded and interpreted the EPR data. K.B.H. participated in all five of the above experiments and recorded and interpreted spectroscopic data. J.F., with K.B.H., recorded and interpreted some of the transient absorption data and all of the re-oxidation data. M.K. helped with the global analysis of the re-oxidation data. M.J.G., V.D., J.R.W. and P.D.F.M. made spectroscopic measurements of magnetic field effects. D.J.C.S. helped to develop the picosecond TA apparatus. J.L. and Y.W. performed spin dynamics calculations. T.L.P. and G.M. reproduced and helped to interpret the EPR data. A.S.G. recorded and interpreted mass spectra. M.B. expressed and purified chicken CRY4. M.H., S.H., G.D. and S.J.K. expressed and purified some of the ErCRY4 protein samples. Y.C., J.S.T. and J.X. expressed and purified pigeon CRY4. H.Y., H.W., K.-W.K., R.B. and C.X. provided advice on protein expression. I.A.S. performed molecular dynamics simulations and provided advice on cryptochrome structure and dynamics. L.E.J. had oversight of the organization and administration of the optical spectroscopy measurements. P.J.H., H.M., C.R.T. and S.R.M. conceived the study. P.J.H., H.M., C.R.T., S.R.M. and C.X. supervised the work. P.J.H. and H.M. wrote the manuscript, and all authors commented on the manuscript.
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Xu, J., Jarocha, L.E., Zollitsch, T. et al. Magnetic sensitivity of cryptochrome 4 from a migratory songbird. Nature 594, 535–540 (2021). https://doi.org/10.1038/s41586-021-03618-9
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DOI: https://doi.org/10.1038/s41586-021-03618-9
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James Hilton
Animals can hear and see things that human beings cannot. This is a fact. If interested, supporting documentation can be offered.
Graham Clayden Replied to James Hilton
How can so many intelligent people get things so totally wrong? Because a few simple observations show that birds in fact navigate in the same way as mariners did in the age of sail. Birds have a biological chronometer and a way of detecting the precise position of the sun in order to ascertain in which direction they should fly, in order to return to a known location. the results from one simple experiment leads me to believe that birds also have a biological altimeter.
Easily replicated experiments, showing known navigational principles are at play, win every time for me, rather than complex and complicated theories . Birds are relatively low in intelligence so their navigational ability can only result from lower brain function, (Maths and calculations are hardly the forte of even accomplished navigators such as homing pigeons.) Keep things simple is my motto, the simplest answer to any problem is usually the correct one.
Roswitha Wiltschko
Comment on the article ’Magnetic
sensitivity of cryptochrome 4 from a migratory bird’by Xu et al. published in Nature 594, p. 535-540
Roswitha Wiltschko and Wolfgang Wiltschko
FB Biowissenschaften, Goethe-Universität
Frankfurt am Main, Germany - e-mail: wiltschko@bio.uni-frankfurt.de
The authors present a thorough in-vitro analysis of the biophysics and biochemistry of cryptochrome 4 (Cry4), focusing on the radical pairs flavin / tryptophan (FADH°/ Trp) formed during photo-reduction, which are found to be highly sensitive to magnetic fields. Although the authors do not explicitly say so, they imply that these are the radical pairs that mediate the birds’ sensitivity to magnetic directions. - However, they do not mention the biological findings that make such a role of the radical pair FADH°/Trp and of Cry4 as receptor molecule rather unlikely.
Fully oxidized cryptochrome requires short-wavelength light from UV to about 500 nm blue to photo-reduce FADox to the radical pair FADH° /Trp (see e.g. 1). Birds, however, are also well oriented in their migratory direction or training direction under narrow band green light (LED lights with peak wavelength of 521, 561, 565 and 571 nm) – that is, wavelengths that cannot photo-reduce FADox to form this radical pair. This was shown for Australian Silvereyes, Zosterops lateralis 2 , European Robins, Erithacus rubecula 3,4, Garden Warbler, Sylvia borin 5, and Zebra Finches, Taeniogygia guttata 6. Yet green light, although not able to photo-reduce fully oxidized FADox, can further reduce the semiquinone FADH° to the fully reduced form FADH-. This, in turn, can be re-oxidized independent of light, forming a second radical pair during re-oxidation (see 1). Thus, as long as there is a certain supply of FADH° and FADH- available, the later part of the photo cycle can run, providing the second radical pair whose nature is not yet entirely clear (see, e.g. 7-11 and others.). Hence the observed orientation under green light which was found to be a temporary phenomenon12 suggests that it is this second radical pair during re-oxidation that is the crucial one for sensing magnetic directions. - Further behavioral experiments showed that the process of magnetic sensing itself does not require light: in a situation with flickering light and a pulsed magnetic field, where light was present without a meaningfulmagnetic information, but the geomagnetic field was present only in total darkness, bird were well orientated 13. This also points to the radical pair formed light-independently during re-oxidation. Light, however, is required to provide the fully reduced form FADH- for re-oxidation 13. Together, these findings clearly indicate that not the radical pair FADH°/Trp formed during photo-reduction, but the one formed during re-oxidation in crucial for sensing magnetic directions.
Another question relates to the nature of the cryptochrome involved. There are four types of cryptochrome found in the avian retina: Cry1a, Cry1b, Cry2 and Cry 4. Cry4 is found in the long-wavelength single cones and double cones14. These cones, however, contain reddish oil droplets15 which absorb the short wavelengths required for photo-reduction of oxidized FADox, so that the location of Cry4 in these cone types makes any role of Cry4 as receptor molecule for magnetic directions rather unlikely. Also, the finding that Cry4 from robins is more sensitive than that of domestic chickens and pigeons does not necessarily argue for Cry4 as receptor molecule for sensing directions. The magnetic compass is by far not restricted to migratory birds, and the two non-migrant species mentioned here have been shown in conditioning experiments to orient by a magnetic compass16,17. So we would not expect them to be less well equipped. Maybe one should think about another function of Cry4 associated with migration?
Altogether, the findings mentioned above clearly speak against a role of Cry4 and the radical
par FADH°/Trp in the avian magnetic compass.
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