Magnetic sensitivity of cryptochrome 4 from a migratory songbird
Authors: Victoire Déjean
Rabea Bartölke
Stefanie J Käsehagen
Stuart R Mackenzie
Glen Dautaj
Ilia A Solov&#x27;yov
Jessica R Walton
Kevin B Henbest
Lauren E Jarocha
Hang Yin
Stefan Weber
Tommy L Pitcher
Jingjing Xu
Haijia Wu
Jiate Luo
Matthew J Golesworthy
Can Xie
Angela S Gehrckens
Marco Bassetto
Daniel J C Sowood
Sabine Richert
Yujing Wei
Marcin Konowalczyk
Maike Herrmann
Simon Horst
Karl-Wilhelm Koch
Jessica Fleming
Gabriel Moise
P J Hore
Henrik Mouritsen
Christiane R Timmel
Joseph S Takahashi
Yogarany Chelliah
Tilo Zollitsch
Jessica Schmidt
Patrick D F Murton
Journal: Nature (2021)
Discipline: Multidisciplinary
Keywords: night-migratory songbirds; magnetic compass; cryptochrome; quantum spin dynamics; photoinduced radical pairs
Affiliations: AG Neurosensory Sciences/Animal Navigation, Institut für Biologie und Umweltwissenschaften, Carl-von-Ossietzky Universität Oldenburg, Oldenburg, Germany., Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, UK., Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, UK., Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany., Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA., Division of Biochemistry, Department of Neuroscience, Carl-von-Ossietzky-Universität Oldenburg, Oldenburg, Germany., Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA., Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA., Research Centre for Neurosensory Science, University of Oldenburg, Oldenburg, Germany., Research Centre for Neurosensory Science, University of Oldenburg, Oldenburg, Germany. ., Department of Physics, Carl-von-Ossietzky Universität Oldenburg, Oldenburg, Germany. ., High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, PR China. ., International Magnetobiology Frontier Research Center, Hefei, PR China. ., Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, UK. ., Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, UK. ., Centre for Advanced Electron Spin Resonance (CAESR), Department of Chemistry, University of Oxford, Oxford, UK. ., AG Neurosensory Sciences/Animal Navigation, Institut für Biologie und Umweltwissenschaften, Carl-von-Ossietzky Universität Oldenburg, Oldenburg, Germany. .

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 birds4-7. 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. 

Retracted: No

PubPeer comments:
 This is a complex paper and I make no claim to understanding the (quantum) chemistry underlying the mechanisms studied in this article. However, despite the magnetic sensitivity of the cryptochrome reactions reported by the authors, I would like to outline what seem to be quantitative challenges before  quantum spin dynamics of photoinduced radical pairs in cryptochrome flavoproteins located in the retinas of the birds can be considered part of a biological compass. The proposed mechanism is summarised in Fig. 4. ![file] Oxidised FAD is photoreduced by blue light and via electron-transfer reactions through the magnetically sensitive grey box produces the potential signalling molecule FADHu2022 (the dot should be superscripted), which can be detected (along with precursor radicals?) in these experiments by changes of absorbance in the 500-600nm range. Fig. 2 shows that its production can be modified by magnetic fields. ![file] Although the difference in absorbance induced by (strong) magnetic fields is quite large when probed using pulsed light (panels a and b), the changes of absorbance with continuous illumination appear to be rather small. Thus, panel c suggests absorbance changes of about 1E-5. I can t find the reference absorbance for these changes (maybe the authors could point to or supply this information?), but given the similarity of conditions to those of other experiments where absolute absorbance is reported, and given that the measurement was performed shortly after switching on the illumination at 450 nm, a baseline absorbance value of 0.01 seems reasonable. The variation induced by the magnetic field would therefore be about 1 part in 1000. (However, maybe part of that background is not due to  potential signalling species?) Quite how an animal might detect the orientation of a magnetic field with such small variations is unclear to me. There is the additional problem that the rate of production of the signalling molecule also depends on light intensity, so the detection of orientation (by moving the head?) must include normalisation for variations in incident light intensity. These birds migrate and presumably navigate at night, when presumably there is less blue light around. This might reduce background radical concentrations, but would presumably also reduce signal? Conversely, most of the cuvette experiments seem to have been carried out under oxidising conditions. Reoxidation of the putative signalling radicals appears to be considerably slower under reducing conditions (hours not minutes; Fig. S18), which might better approximate conditions within cells. Would this not increase background and reduce sensitivity? The magnetic fields used in these experiments have half-maximal effects at around 5mT (e.g. Fig. 2b), which is about 100x the strength of the terrestrial magnetic field of 50u00b5T. The true signals would therefore be at most a few percent of those shown, so the animals would need to detect better than 1 part in 10000. Furthermore, all of the curves (such as in Fig. 2a) where magnetic field was varied are fitted with sigmoids. The authors call these  Lorentzians , these are presumably very standard, because no formula is given or justified. Anyway, if there is an expectation (theoretical and or mechanistic) for this form of curve, the extrapolation of signal to the terrestrial field will be even less favourable (maybe quadratic or exponential rather than linear). All of the experiments are carried out at low temperatures near freezing. In a footnote in the supplementary information (p31), the authors state:  Lowering the temperature and addition of glycerol were found to enhance the magnetic field effects. One would have liked more information about the strength of effects at physiological temperatures. The authors attempt to correct for the effect of temperature on the protonation of the signalling chain with compensating pH changes, but I couldn t find any information about any other aspects of the reaction that might be sensitive to the temperature. , 
 #1 You make a number of good points. If more was known about how magnetoreception works in vivo, I would be able to offer more helpful and detailed responses. It is important to remember that the conditions of our experiments are far from those that exist in cells in the birdsu2019 retinas (lower than physiological temperature, no immobilisation or alignment of the proteins, no interactions with signalling partners, no biochemical amplification, different redox environment). There are reasons for thinking that the magnetic field effects could be larger in vivo. It may not be necessary for photo-reduced proteins to be re-oxidised within minutes in vivo during the night. It is possible that having been used once, a protein molecule is degraded and replaced by freshly expressed protein containing fully oxidised FAD. We have suggested a way in which the u201cnormalisation for variations in incident light intensityu201d could occur: Worster et al., J. R. Soc. Interface 14 (2017) 20170405. If electron spin relaxation is slower in vivo than in vitro, then instead of an approximately Lorentzian dependence on the field strength, there should be a so-called low field effect which causes the response to be roughly linear in field strength for weak ( 1 mT) magnetic fields. See, for example, Fig. 6 of: Maeda et al., Proc. Natl. Acad. Sci. USA, 109 (2012) 4774-4779. As you have probably guessed, we did the experiments at reduced temperatures to increase the size of the magnetic field effects. The origins of the temperature dependence are not clear u2013 spin relaxation in the radical pair is probably a factor. There is a frustratingly large number of things we simply donu2019t know or donu2019t understand. Further experiments will no doubt show whether Cry4 is the right molecule and whether radical pairs are the right mechanism. , 
 #3 Dear *Bulbophyllum Restrepia*, I think your estimate of 1 part in 100,000 is a bit pessimistic. As I said in response to your earlier questions, itu2019s difficult to extrapolate from our *in vitro* conditions to the imperfectly understood but undoubtedly very different conditions inside photoreceptor cells in a birdu2019s eye. Nevertheless, even if the magnetic field effects *in vivo* are nearer 1 part in 100 or 1000, youu2019re right that the cancellation of light intensity effects would have to be very precise. Apart from the figure in your letter, Iu2019m afraid we havenu2019t paid much attention to temperature. There are so many differences between the conditions *in vitro* and those expected *in vivo* that it didnu2019t seem a priority to focus on temperature. With regard to the Lorentzian form, we used the following expression for the smooth lines in the plots of magnetic field effects: ![file] *B* is the strength of the applied magnetic field. *a* ( 0) is the limiting magnetic field effect at high field. *b* is the field strength at which the magnetic field effect equals 0.5*a*: this is the parameter we called *B*_1 2 in the article. Youu2019re right that this expression is empirical: under most, but not all, conditions the measured and calculated magnetic field effects have an approximately Lorentzian dependence on field strength. In case youu2019re interested, Wikipedia has an article on the Lorentz distribution, also known as the [Cauchy distribution] . Best wishes, Peter Hore P.S. *Bulbophyllum Restrepia*, I am curious to know who you are. You could send me a separate email if you wish to remain anonymous on-line. , 
 Dear Dr Hore, Thank you again for the clarifications. The magnetic sensitivity of the reaction (with pulsed light) impresses me, even if the manner of its possible exploitation remains quite unclear for now. Could I ask one further favour of you or maybe of your collaborators? To identify what you believe is the paper providing the strongest demonstration of the existence of a magnetic sense in any animal? (I.e. I m not counting magnetic bacteria.) Please note that I m seeking a single article; I don t want a  lots of papers list, just the very best. , 
 #5 Dear Bulbophyllum Restrepia, u201cThe paper providing the strongest demonstration of the existence of a magnetic sense in any animal?u201d I suppose it would have to be one of the early papers by Friedrich Merkel, Wolfgang Wiltschko and Roswitha Wiltschko in the late 1960s and early 1970s. The first of these in English seems to be: W. Wiltschko and R. Wiltschko (1972).  Magnetic compass of European robins. Science 176, 62-64. I believe this work was the first to show convincingly that migratory songbirds can sense the direction of the geomagnetic field. In case you are interested, there are two earlier articles: W. Wiltschko and F. W. Merkel (1966).  Orientierung zugunruhiger Rotkehlchen im statischen Magnetfeld. Verh. der Deutsch. Zoolog. Ges. 59: 362-367. W. Wiltschko (1968).  u00dcber den Einfluu00df statischer Magnetfelder auf die Zugorientierung der Rotkehlchen (Erithacus rubecula). Z. Tierpsychol. 25: 537-558. Best wishes, Peter Hore P.S. Iu2019m still curious to know who you are. Why hide behind an orchid? , 
 Could authors comment on purity of their site-specific mutants of ErCRY4? SDSu2013PAGE Coomassie blue staining Figure 1b highlights multiple bands for the same lane. It is more obvious upon brightness contrast enhancement. Could authors provide the original gel image? ![image] , 
 #7 Dear Eleutherodactylus Thorectes, The original gel image can be found on page 4 of the Methods and Supplementary Information PDF. I have asked my colleagues to reply to your question about the purity of the samples. Best wishes, Peter Hore , 
 Dear Dr Hore, Thank you for your quick reply. For the convenience of other readers, below I link directly to the two papers you mention. https: royalsocietypublishing.org doi 10.1098 rsif.2017.0405 https: www.pnas.org content 109 13 4774 In the first article, you state (abstract)  Here we propose a simple solution in which these extraneous effects are cancelled by taking the ratio of the signals from two neighbouring populations of magnetoreceptors. I realise that some sort of ratio or subtraction might be one possible mechanism (the additional problem of polarisation, which is the main subject of that paper, hadn t occurred to me). As you suggest, the ratio subtraction could be calculated instantaneously with  crossed receptors. Yet that ratio or subtraction would still need to be spectacularly precise. It doesn t seem at all simple to imagine how a biochemical system could be set up to attain the precision of 1 part in 100000 (assuming my ballpark estimates above were not too far wrong). Regarding temperature effects, it is disappointing that no quantitative information was given. For instance, was the signal 3 times smaller at raised temperature or undetectable? Fig. 5B from the PNAS paper shows the temperature dependence of the magnetic field effect with a less sensitive protein. It s a little difficult to extrapolate using the colour key, but a simple-minded approach suggests that the signal would be less than zero at 40u00b0C. Was a similar dependence observed in these experiments? ![imge] A sign of my ignorance, I have been unable to discover what the (general) formula for your  Lorentzian curve is. Could you maybe give it or indicate a reference? If I understand your comment correctly, it is expected theoretically under some conditions but may not always apply. , 
 #7 Dear Eleutherodactylus Thorectes, Thanks for your comments. As Prof. Hore said, you should be able to find the original gel figure in the Methods and supplementary information. All the ErCry4 protein was recombinantly expressed in E.coli cell culture and then extracted and purified in native condition from numerous pooled E.coli expressed proteins. No single technique is 100% perfect. Of course, the purification does not result in 100% pure target protein. But our samples are very clean for the used methodology. Considering the percentage of the u2018impurityu2019 in the protein sample is very small, we are very confident that the tiny amount of impurity will not affect the overall magnetic field effect we measured, as the impurities are very unlikely to bind FAD. Best wishes, Jingjing Xu ,