Biology subjects

Mouritsen, H.

Publications and source records attributed to Mouritsen, H..

17 recordsLinked to original sources

bioRxiv2026

Expression patterns and interaction profiles of heterotrimeric transducin subunits in the retina of the European robin ( Erithacus rubecula )

The heterotrimeric G-protein transducin (Gt) is mediating phototransduction in rod and cone cells of the vertebrate retina, but its expression patterns in migratory songbirds is unknown. We characterised Gt expression in the European robin, a night-migratory songbird known for its light-dependent magnetoreception. One well-supported hypothesis of magnetoreception involves radical-pair formation in the blue light receptor cryptochrome type 4a. The - and {gamma}-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 by combining single cell RNA sequencing and immunohistochemistry. Protein-protein interaction was tested by pulldown, co-immunoprecipitation, and NanoBiT luminescence assays. G-protein subunits Gt{beta}1 and Gt{beta}3 are predominantly expressed in rods and cones, and Gt{gamma}T2 was the principal isoform in cones, whereas Gt{gamma}11 was associated with rods. In contrast, we did not detect Gt{gamma}10 expression in either photoreceptor type. Interaction assays demonstrated that all three {beta}{gamma} combinations; {beta}{gamma}T2, {beta}{gamma}10, and {beta}{gamma}11, can associate in vitro. These findings indicate that {beta}{gamma} dimer formation in vivo is likely constrained by the photoreceptor-specific expression of the respective subunits. The absence of Gt{gamma}10 expression in rods and cones does not support a role in photoreceptor-based magnetoreception.

Molecular Biology↗

bioRxiv2026

Non-equilibrium vibrational dynamics govern ultrafast electron transfer in avian cryptochrome

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.

biophysics↗

bioRxiv2026

Magnetic compass orientation behaviour of Eurasian blackcaps at the predicted 110-120 MHz upper cut-off frequency for radiofrequency-field effects

Night-migratory songbirds use the Earths 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{degrees} when the field was rotated 120{degrees} 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).

animal behavior and cognition↗

bioRxiv2026

The Role of Magnetic and Celestial Cues in Orientation and Navigation of Red Underwing (Catocala nupta), a European Migratory Moth

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 Earths 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.

animal behavior and cognition↗

bioRxiv2026

Magnetic sensitivity of cryptochrome 4a in domesticated quail with migratory origins

Magnetoreception, the ability of animals to sense the Earths 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.

biophysics↗

bioRxiv2026

Absence of 8-HDF and MTHF Antenna Chromophore Binding in ErCRY4a Suggests a Possible Flavin-Only Cofactor State: Insights from Biochemical and Computational Analyses

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 (ErCRY4a) also binds 8-HDF and/or MTHF. 8-HDF binding was studied by co-expressing ErCRY4a 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 (Xl6-4PL) which is known to bind both FAD and 8-HDF. This experiment resulted in successful binding of 8-HDF to Xl6-4PL, but not to ErCRY4a. 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 ErCRY4a. To theoretically understand the binding of potential antenna chromophores to ErCRY4a, we performed computational analyses. We found no similarity at the relevant binding sites between the sequences of ErCRY4a with proteins shown to bind MTHF or 8-HDF. This suggests that the binding pocket is not conserved. Our study proposes that ErCRY4a only harbor one light-sensitive cofactor, which in turn suggests a functional specialization different from most photolyases.

biochemistry↗

bioRxiv2025

A Charge-reversal Point Mutation Completely Depletes Flavin Chromophore from European Robin Cryptochrome 4a Protein

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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/690116v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@93344forg.highwire.dtl.DTLVardef@4f642corg.highwire.dtl.DTLVardef@39912corg.highwire.dtl.DTLVardef@169e1de_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Mapping the magnetoreceptive brain: A 3D digital atlas of the migratory bird Eurasian blackcap (Sylvia atricapilla)

Birds undisputedly range amongst natures foremost navigators. To successfully navigate between breeding and wintering quarters, they, in addition to other natural orientation cues, rely on their ability to sense the Earths magnetic field. For this reason, migratory birds have become key model species for studying the sensory mechanisms underlying magnetic field-guided navigation, as evidenced by the identification of several brain regions believed to be involved in processing magnetic field information. However, there is as yet no readily accessible, high-resolution three-dimensional (3D) brain atlas to serve as a common reference within and across studies. Here we provide the neuroscience research community with the first freely available, digital, high-resolution (25 {micro}m), 3D bird brain atlas. It is based on light microscopy images from ten Eurasian blackcaps (Sylvia atricapilla), a night-migratory songbird widely used model species in magnetoreception and navigation research. We outline the individual steps for the creation of a brain atlas, from whole-brain imaging using serial-section, two-photon tomography, to the creation of an average template at an isotropic 25-{micro}m voxel size, and finally to brain area segmentation and annotation. In this first version of the atlas, we have mapped a total of 24 brain areas, including 6 principal compartments, 13 conspicuous anatomical subdivisions common to all bird species and 5 functionally defined areas of the visual and trigeminal sensory systems involved in processing magnetic field information. This atlas is accessible via the standardised BrainGlobe Atlas API, making it compatible with a growing suite of computational neuroanatomy tools provided by the BrainGlobe Initiative. This integration enables precise alignment of future experimental data to a common coordinate space, facilitating collaboration, data visualization and sharing. Furthermore, this resource enables the accurate localization and comparison of implanted devices, injection sites, and/or cell populations across individual brains, both within and across studies.

neuroscience↗

bioRxiv2025

Cryptochrome 4b protein is likely irrelevant for the radical pair based magnetoreception in the European robin

Avian cryptochrome 4 (Cry4) protein is a putative magnetosensitive molecule facilitating precise long-distance navigation in migratory birds. Two splice variants of Cry4 were reported in European robin (Erithacus rubecula), namely ErCry4a and ErCry4b. It is known that ErCry4a protein exhibits electron transfer between the flavin adenine dinucleotide (FAD) cofactor and tryptophan residues that generates magnetically sensitive radical pairs for magnetoreception. However, little is known about the ErCry4b isoform. We therefore characterized the properties of ErCry4b to see whether it fulfills prerequisites to be a radical pair magnetic sensor molecule. Our results show that ErCry4b protein does not bind FAD in vitro. Computational structure simulations revealed that the FAD non-binding in ErCry4b is likely due to protein structure dynamics. Furthermore, ErCry4b protein abundance in the robin retina, cerebellum and liver is below the detection limit of immunoprecipitation assays coupled with mass spectrometry. Meanwhile, transcript analyses show that ErCRY4b mRNA abundance is 10 times less than ErCRY4b in the retina. In conclusion, ErCry4b does not fulfill the prerequisites to be a radical pair based magnetic sensing molecule due to the lack of FAD binding, and it might not even be expressed as a functional protein in the European robin.

biochemistry↗

bioRxiv2025

Directionality range in Emlen funnels

Emlen funnels can be used to study the birds ability to orient during the migratory seasons. Birds are so eager to migrate that they will jump in the direction in which they want to fly, even if they are placed in small cages during the night. Emlen funnels have therefore been used for decades to study the sensory capabilities and mechanisms that migratory birds use to find their way. A significant part of this research has focused on how night-migratory songbirds perceive the Earths magnetic field. Even though Emlen funnels have been proven quite successful in capturing the birds behavioural responses to different experimental conditions, the orientation behaviour of night-migratory songbirds in Emlen funnels is very noisy, i.e. tends to have a low signal to noise ratio. This noise makes Emlen funnel experiments very time consuming and limits the types of questions that can be studied to those which require very few different experimental conditions (permutations). Furthermore, the experimental design choices can be crucial, e.g. degree of blinding of the experimenters and data evaluators to the conditions tested, pre-testing of birds to make sure they are in a migratory state, and planning of the effective sample sizes. Hence, different traditions in experimental design choices can reduce reproducibility and comparability and render the interpretation of the results non-trivial. To better understand Emlen funnel data and the minimal requirements for good experimental design, we constructed and analyzed a large data set that we compiled by combining behavioural data from many previous Emlen funnel studies performed in Oldenburg. Our results provide realistic ranges for the expected orientation of the birds in Emlen funnels, which can be useful (and in some research crucial) for predicting the optimal sample sizes for future experiments. Our results thus offer concrete information for the design and analysis of statistically powerful future magnetic orientation experiments.

animal behavior and cognition↗

bioRxiv2025

Morphology and connectivity of retinal horizontal cells in two avian species

In the outer vertebrate retina, the visual signal is separated into intensity and wavelength information. In birds, seven types of photoreceptors (one rod, four single cones, and two members of the double cone) mediate signals to >20 types of second-order neurons, the bipolar cells and horizontal cells. Horizontal cells contribute to color and contrast processing by providing feedback signals to photoreceptors and feedforward signals to bipolar cells. In fish, reptiles, and amphibians they either encode intensity or show color-opponent responses. Yet, for the bird retina, the number of horizontal cell types is not fully resolved and even more importantly, the synapses between photoreceptors and horizontal cells have never been quantified for any bird species. With a combination of light microscopy and serial EM reconstructions, we found four different types of horizontal cells in two distantly related species, the domestic chicken and the European robin. In agreement with some earlier studies, we confirmed two highly abundant cell types (H1, H2) and two rare cell types (H3, H4), of which H1 is an axon-bearing cell, whereas H2-H4 are axonless. Horizontal cell contacts to photoreceptors were type-specific and similar to the turtle retina, which confirms the high degree of evolutionary conservation in the vertebrate outer retina. Our data further suggests that H1 and potentially H2 cells may encode intensity, whereas H3 and H4 may represent color opponent horizontal cells which may contribute to the birds superb color and/or high acuity vision.

neuroscience↗

bioRxiv2024

Full-length Cryptochrome 1 in the outer segments of the retinal blue cone photoreceptors in humans and great apes suggests a role beyond transcriptional repression

Mammalian cryptochrome 1 (CRY1) is a central player in the circadian transcription-translation feedback loop, crucial for maintaining a roughly 24-hour rhythm. CRY1 was suggested to also function as blue-light photoreceptor in humans and has been found to be expressed at the mRNA level in various cell types of the inner retina. However, attempts to detect CRY1 at the protein level in the human retina have remained unsuccessful so far. Using various C-terminal specific antibodies recognizing full-length CRY1 protein, we consistently detected selective labelling in the outer segments of short wavelength-sensitive (SWS1, "blue") cone photoreceptor cells across human, bonobo, and gorilla retinae. No other retinal cell types were stained, which is in contrast to what would be expected of a ubiquitous clock protein. Subcellular fractionation experiments in transfected HEK cells using a C-terminal specific antibody located full-length CRY1 in the cytosol and membrane fractions. Our findings indicate that human CRY1 has several different functions including at least one non-clock function. Our results also raise the likely possibility that several different versions of CRY1 exists in humans. We suggest that truncation of the C-terminal tail, maybe to different degrees, may affect the localization and function of human CRY1.

cell biology↗

bioRxiv2024

Comparison of retinol binding protein 1 with cone specific G-protein as putative effector molecules in cryptochrome signalling

Vision and magnetoreception in navigating songbirds are strongly connected as recent findings link a light dependent radical-pair mechanism in cryptochrome proteins to signalling pathways in cone photoreceptor cells. A previous yeast-two-hybrid screening approach identified six putative candidate proteins showing binding to cryptochrome type 4a. So far, only the interaction of the cone specific G-protein transducin -subunit was investigated in more detail. In the present study, we compare the binding features of the G-protein -subunit with those of another candidate from the yeast-two-hybrid screen, cellular retinol binding protein. Purified recombinant European robin retinol binding protein bound retinol with high affinity, displaying an EC50 of less than 5 nM, thereby demonstrating its functional state. We applied surface plasmon resonance and a Forster resonance transfer analysis to test for interactions between retinol binding protein and cryptochrome 4a. In the absence of retinol, we observed no robust binding events, which contrasts the strong interaction we observed between cryptochrome 4a and the G-protein -subunit. We conclude that retinol binding protein is unlikely to be involved in the primary magnetosensory signalling cascade.

biochemistry↗

bioRxiv2024

European Robin Cryptochrome-4a Associates with Lipid Bilayers in an Ordered Manner, Fulfilling a Molecular-Level Condition for Magnetoreception

Since the middle of the 20th century, long-distance avian migration has been known to rely partly on the geomagnetic field. However, the underlying sensory mechanism is still not fully understood. Cryptochrome 4a (ErCry4a), found in European Robin (Erithacus rubecula), a night-migratory songbird has been suggested to be a magnetic sensory molecule. It is sensitive to external magnetic fields via the so-called radical-pair mechanism. ErCry4a is primarily located in the outer segments of the double cone photoreceptor cells in the eye, which contain stacked and highly ordered membranes that could facilitate the anisotropic attachment of ErCry4a needed for magnetic compass sensing. Here, we investigate possible interactions of ErCry4a with a model membrane that mimics the lipid composition of outer segments of vertebrate photoreceptor cells by using experimental and computational approaches. Experimental results show that the attachment of ErCry4a to the membrane could be controlled by the physical state of lipid molecules (average area per lipid) in the outer leaflet of the lipid bilayer. Furthermore, polarization modulation infrared reflection absorption spectroscopy allowed us to determine the conformation, motional freedom, and average orientation of the - helices in ErCry4a in a membrane-associated state. Atomistic molecular dynamics studies supported the experimental results. A [~]1000 kcal mol-1 decrease in the interaction energy as a result of ErCry4a membrane binding was determined compared to cases where no protein binding to the membrane occurred. At the molecular level, the binding seems to involve negatively charged carboxylate groups of the phosphoserine lipids and the C-terminal residues of ErCry4a. Our study reveals a potential direct interaction of ErCry4a with the lipid membrane and discusses how this binding could be an essential step for ErCry4a to propagate a magnetic signal further and thus fulfill a role as a magnetoreceptor.

biophysics↗

bioRxiv2023

Dimerisation of European robin cryptochrome 4a

Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerisation of avian cryptochromes and the role it could play in the mechanism of magnetic sensing in migratory birds. Here we present a combined experimental and computational investigation of the dimerisation of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulphide bonds, chemical cross-linking and photometric measurements show that disulphide-linked dimers are routinely formed, the most likely cysteines being C317 and C412. Computational modelling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.

biochemistry↗

bioRxiv2023

Evidence for adaptive evolution towards high magnetic sensitivity of potential magnetoreceptor in songbirds

Migratory birds possess remarkable accuracy in orientation and navigation, which involves various compass systems including the magnetic compass. Identifying the primary magnetosensor remains a fundamental open question. Cryptochromes (Cry) have been shown to be magnetically sensitive, specifically Cry4 shows enhanced magnetic sensitivity in migratory songbirds compared to resident species. Here, we investigate cryptochromes and their potential involvement in magnetoreception in a phylogenetic framework, integrating molecular evolutionary analyses with protein dynamics modeling. We base our analysis on 363 bird genomes and associate different selection regimes with migratory behaviour. We show that Cry4 is characterized by strong positive selection and high variability, typical characteristics of sensor proteins. We identify key sites that likely facilitated the evolution of a highly optimized sensory protein for night time compass orientation in songbirds and a potential functional shift or specialisation. Additionally, we show that Cry4 was lost in hummingbirds, parrots and Tyranni (Suboscines) and thus identified a natural comparative gene knockout, which can be used to test the function of Cry4 in birds. In contrast, the other two cryptochromes Cry1 and Cry2, were highly conserved in all species, indicating basal, non-sensory functions. Our results strengthen the hypothesised role of Cry4 as sensor protein in (night)-migratory songbirds.

evolutionary biology↗

bioRxiv2016

A magnetic protein compass ?

Qin et al. (A magnetic protein compass, Nature Materials 15, 217-226, 2016) claim that \"MagR is the first known protein that carries an intrinsic magnetic moment at ambient temperature\". We show here that the claim must, unfortunately, be fundamentally wrong.

biophysics↗