Localisation of cryptochrome 2 in the avian retina.

Cryptochromes are photolyase-related blue-light receptors acting as core components of the mammalian circadian clock in the cell nuclei. One or more members of the cryptochrome protein family are also assumed to play a role in avian magnetoreception, but the primary sensory molecule in the retina of migratory birds that mediates light-dependent magnetic compass orientation has still not been identified. The mRNA of cryptochrome 2 (Cry2) has been reported to be located in the cell nuclei of the retina, but Cry2 localisation has not yet been demonstrated at the protein level. Here, we provide evidence that Cry2 protein is located in the photoreceptor inner segments, the outer nuclear layer, the inner nuclear layer and the ganglion cell layer in the retina of night-migratory European robins, homing pigeons and domestic chickens. At the subcellular level, we find Cry2 both in the cytoplasm and the nucleus of cells residing in these layers. This broad nucleic expression rather points to a role for avian Cry2 in the circadian clock and is consistent with a function as a transcription factor, analogous to mammalian Cry2, and speaks against an involvement in magnetoreception

Tracking the Electron Transfer Cascade in European Robin Cryptochrome 4 Mutants

The primary step in the elusive ability of migratory birds to sense weak Earth-strength magnetic fields is supposedly the light-induced formation of a long-lived, magnetically sensitive radical pair inside a cryptochrome flavoprotein located in the retina of these birds. Blue light absorption by a flavin chromophore triggers a series of sequential electron transfer steps across a tetradic tryptophan chain towards the flavin acceptor. The recent ability to express cryptochrome 4 from the night-migratory European robin (Erithacus rubecula), ErCry4, and to replace the tryptophan residues individually by a redox-inactive phenylalanine offers the prospect of exploring the role of each of the tryptophan residues in the electron transfer chain. Here, we compare ultrafast transient absorption spectroscopy of wild type ErCry4 and four of its mutants having phenylalanine residues in different positions of the chain. In the mutants we observe that each of the first three tryptophan residues in the chain adds a distinct relaxation component (time constants 0.5, 30 and 150 ps) to the transient absorption data. The dynamics in the mutant with a terminal phenylalanine residue are very similar to those in wild type ErCry4, excepted for a reduced concentration of long-lived radical pairs. The experimental results are evaluated and discussed in connection with Marcus-Hopfield theory, providing a complete microscopic insight into the sequential electron transfers across the tryptophan chain. Our results offer a path to studying spin transport and dynamical spin correlations in flavoprotein radical pairs