Henrik Mouritsen

The heterotrimeric G-protein transducin (Gt) is among the key proteins mediating phototransduction in rod and cone cells of the vertebrate retina. Even though this protein has been extensively characterized in mammals, little is known about its expression patterns in migratory songbirds. Here we characterised Gt expression in the European robin, a night-migratory songbird known for its light-dependent magnetoreception. The mechanism underlying magnetoreception is not fully understood, but one well-supported hypothesis involves a radical-pair formation in the blue light receptor cryptochrome type 4a. The α- and γ-subunits of cone specific transducin have been identified as possible interaction partners of cryptochrome 4a. Therefore, we analysed the expression patterns of various G-protein subunits in bird photoreceptors. Specifically, we combined single cell RNA sequencing and immunohistochemistry, and tested for protein interaction by pulldown, co-immunoprecipitation, and NanoBiT luminescence assays. We show that genes for G-protein subunits GNB1 and GNB3 (coding for Gtβ1 and Gtβ3, respectively) are predominantly expressed in rods and cones. Among γ-subunits, GNGT2 (coding for GtγT2) was the principal isoform in cones, whereas GNG11 (coding for Gtγ11) was associated with rods. In contrast, we did not detect GNG10 (coding for Gtγ10) expression in either photoreceptor type. Interaction assays demonstrated that all three βγ combinations; βγT2, βγ10, and βγ11, can associate in vitro . These findings indicate that βγ dimer formation in vivo is likely constrained by the photoreceptor-specific expression of the respective subunits. Furthermore, the absence of GNG10 expression in rods and cones does not support a role of this γ-subunit in photoreceptor-based magnetoreception.

Photoactivated intermolecular electron transfer (ET) in cryptochromes proceeds along chains of aromatic residues and creates a spatially separated pair of radical electrons. Ultrafast time-dependent spectroscopy can provide experimental insight into this process and theoretical estimates of charge transfer rates are commonly obtained via Marcus theory. Here, we present a new perspective on the ET in European robin cryptochrome 4a (ErCry4a) that synthesizes insights from real-time ET calculations, ultrafast spectroscopic measurements and analytical derivations. The simulations exemplify that molecular vibrations play an essential role in enabling the ET dynamics, which was further rationalized through analytical derivations. Ultrafast pump-probe spectroscopy provided experimental access to the first 1.5 ns of the ET cascade, where multiple radical pair recombination rates arise due to the dynamic equilibrium along the ET chain. We show that the motions of the protein environment and the ET dynamics are inseparably coupled, violating the timescale separation required for Marcus theory. The presented results highlight that non-equilibrium coupling between electronic and nuclear motion dominates ET kinetics in ErCry4a during the first nanosecond after photo-excitation. The findings exemplify the limits of Marcus theory and refine the interpretation of ultrafast spectroscopic signatures in cryptochromes.

Night-migratory songbirds use the Earth’s magnetic field to guide their migratory journeys. Most evidence suggests that the magnetic compass sensor is a flavin-tryptophan radical pair whose operation is disrupted by broadband radiofrequency (RF) fields at frequencies up to ∼116 MHz. Here, we test whether broadband 110–120 MHz RF fields affect the magnetic orientation behaviour of night-migratory Eurasian blackcaps ( Sylvia atricapilla ). We found that the birds oriented in their expected migratory direction in the natural geomagnetic field (NMF) control condition and that they turned their orientation ∼120° when the field was rotated 120° horizontally (changed magnetic field, CMF). When they were exposed to broadband 110–120 MHz RF fields, the birds continued to orient in the appropriate direction in the NMF. In the CMF, we found orientation behaviour that was not consistent with the expected direction. We conclude that the birds could still orient when exposed to 110–120 MHz fields, but speculate that their magnetic orientation capabilities might have been somewhat reduced compared to the control condition. We suggest that 110–120 MHz could represent a grey zone of reduced magnetic orientation capability centred at the cut-off frequency predicted for a flavin-based radical pair (probably in the flavoprotein cryptochrome).

Bogong moths (Agrotis infusa) migrate biannually up to 1,000 km between their breeding areas and geographically restricted aestivation sites in the Australian Alps ^1,2 . These moths navigate towards their distant goal using the stars as a compass ³ but navigate correctly even when the stars are obscured by clouds ³ , suggesting that their magnetic sense ⁴ likely harnesses the Earth’s magnetic field as a compass. However, the nature of this compass, and of the magnetic sense underlying it, remain unknown. Here we show that Bogong moths, tethered in a flight arena under a full-spectrum randomised starry night sky, navigate in their inherited migratory direction using the geomagnetic field alone. If both field inclination and polarity are turned by 180°, or if inclination is turned alone, moths fly in the opposite direction. If polarity is turned alone, moths fail to turn. Furthermore, if violet-blue light (380 nm < λ < 500 nm) is removed from the illumination spectrum, or if tethered moths are exposed to weak broadband radiofrequency fields (150 kHz – 60 MHz), magnetic orientation is abolished. Our results indicate that Bogong moths use the geomagnetic field as an inclination compass for long-distance navigation at night and possess a light-dependent radical-pair-based magnetic sense ⁵ .

Nocturnal migration is a remarkable phenomenon observed in many insect species, including moths. Migratory moths are capable of maintaining precise directional orientation during migration, as demonstrated in both laboratory and field studies, suggesting that they use multiple environmental cues for orientation and navigation. Recent studies on Australian Bogong moths revealed that these animals can use stellar cues and likely the geomagnetic field (in conjunction with local visual cues) to select and maintain population-specific migratory direction. However, the underlying orientation mechanisms used by most other migratory moths are still largely unresolved. Further, it remains unclear whether migratory moths can adjust their orientation using Earth’s magnetic field parameters for determining their position relative to the goal (i.e. location or map information) – an ability clearly shown in some migratory birds which respond to virtual magnetic displacements by correcting their orientation (experiments when animals are exposed to magnetic cues corresponding to other geographic locations). Here, we present results from virtual magnetic displacement experiments conducted on red underwings ( Catocala nupta ). In addition, we tested their orientation under simulated overcast conditions and in a vertical magnetic field to get indications whether this species relies on geomagnetic or celestial cues to maintain its population-specific migratory direction. Our results show that (1) red underwings did not compensate for virtual magnetic displacement, indicating the absence of a magnetic map; (2) they remained significantly oriented in the absence of geomagnetic information, suggesting the use of a stellar compass; and (3) there was no evidence of magnetic compass orientation in absence of any visual cues.

Magnetoreception, the ability of animals to sense the Earth’s magnetic field, is a fascinating biological phenomenon. Cryptochromes, in particular cryptochrome 4a (CRY4a), have emerged as potential key players in mediating magnetic sensing in various bird species. Building on an earlier investigation of magnetic field effects on European robin ( Erithacus rubecula ) CRY4a, we focus here on CRY4a from the common/Japanese quail ( Coturnix coturnix/japonica ). Japanese quail is one of the very small number of domesticated bird species whose wild forms are migratory. A detailed spectroscopic study of purified quail CRY4a shows that it has magnetic properties similar to robin CRY4a, suggesting that the quail could be a promising additional experimental model with which to unravel the intricacies of magnetoreception in migratory birds.

Cryptochromes and photolyases are blue-light-sensitive flavoproteins that generally bind flavin adenine dinucleotide (FAD) and have distinct functions. Cryptochrome 4a (CRY4a) is a protein expressed in the double-cone photoreceptors of the retina in migratory songbirds like European robin ( Erithacus rubecula ) and is hypothesized as the primary sensor for avian magnetoreception. In addition to FAD, most photolyases and some cryptochromes bind antenna chromophores such as 8-hydroxy-5-deazaflavin (8-HDF) or 5,10-methenyltetrahydrofolate (MTHF) to enhance light absorption. Here, we investigated whether Erithacus rubecula Cryptochrome 4a ( Er CRY4a) also binds 8-HDF and/or MTHF. 8-HDF binding was studied by co-expressing Er CRY4a with the fbIC gene that encodes for 8-HDF synthase and thus for production of 8-HDF in E. coli . As a positive control for 8-HDF binding, we expressed Xenopus laevis 6-4 photolyase ( Xl 6-4PL) which is known to bind both FAD and 8-HDF. This experiment resulted in successful binding of 8-HDF to Xl 6-4PL, but not to Er CRY4a. We studied the binding of MTHF using in vitro reconstitution followed by UV-Vis spectroscopy and isothermal titration calorimetry (ITC) assays. No interaction was observed between MTHF and Er CRY4a. To theoretically understand the binding of potential antenna chromophores to Er CRY4a, we performed computational analyses. We found no similarity at the relevant binding sites between the sequences of Er CRY4a with proteins shown to bind MTHF or 8-HDF. This suggests that the binding pocket is not conserved. Our study proposes that Er CRY4a only harbor one light-sensitive cofactor, which in turn suggests a functional specialization different from most photolyases.

Our understanding of bird orientation guided by magnetic and visual cues is primarily based on Emlen funnel experiments. Migration-motivated birds jump in the direction they want to fly, and their feet leave marks on paper lining the funnel, which yields the preferred direction. Despite the low signal-to-noise ratio, this paradigm has proven instrumental for studying magnetoreception in birds. However, the high noise limits the questions that can be answered and there is no data-informed guideline for selecting sample sizes that have a high likelihood to be conclusive. Furthermore, differences in experimental design traditions limit comparison and reproducibility across studies, slowing down discovery. We performed a large meta-analysis across double-blind magnetic orientation studies with Emlen funnels performed at Oldenburg to statistically characterize Emlen funnel data and determine minimal sampling requirements for conclusive experimental design. The analysis confirms that pre-selecting migration-motivated animals before the real experiments start improves statistical power by reducing noise. We also highlight mathematical limitations of the widely used directionality measure ‘r’, due to lacking sample-size bias correction, and present realistic ranges for expected bird directedness in Emlen funnels. Combined, these results provide critical design and analysis guidelines for statistically informative magnetic orientation experiments.

Neural tissues are exceptionally sensitive to oxygen deprivation and rely on a dense network of blood vessels to support their extraordinarily high metabolic demands for oxygen, nutrients and clearance of waste products^1,2,3,4. In birds, one of the metabolically most demanding neural tissue—the retina—lacks internal blood vessels^5,6. This raises the question of how such a metabolically demanding neural tissue can function without blood perfusion. Here we show that, while the photoreceptor outer segments in the outer retina have access to oxygen, the inner bird retina operates under chronic anoxia, supported by anaerobic glycolysis in the retinal neurons. We provide evidence that the pecten oculi—a uniquely vascularized structure in the vitreous humour of birds, the function of which has been debated for centuries^5,6,7,8,9—supplies the anoxic inner retina with glucose and removes lactic acid. We suggest that the pecten’s metabolic support of the bird retina’s anoxia tolerance enabled first the evolution of a thick cell-dense, avascular retina, which secondarily served as an exaptation enabling retinal function during high-altitude migrations.

Cryptochrome 4a (Cry4a) is a magnetically sensitive protein that could enable night-migratory birds to sense the geomagnetic field for navigation. The key to the protein magnetic sensitivity is the flavin adenine dinucleotide (FAD) cofactor, which initiates the electron transfer within the protein leading to a spin-correlated radical pair. De-spite its importance, the mechanism of FAD binding in avian Cry4a proteins remains unclear. Here we show that point mutagenesis of positively charged arginine residue at position 356 to negatively charged glutamic acid completely depletes FAD binding from European robin ( Erithacus rubecula ) Cry4a. The result indicates that electrostatic in-teractions constitute the primary driving force that enables FAD binding in European robin Cry4a. The finding provides new structural insight into the molecular basis of FAD binding in Cry4 and advances our understanding on the biophysical underpinnings of bird magnetoreception. Table of Contents Graphic: