Cry1a Cryptochrome has the Characteristics of a Receptor for Avian Magnetosensing

Abstract

Cryptochromes are blue light receptors found in plants and animals which have been linked to magnetosensitivity in multiple organisms. Evidence from migratory birds has shown that the avian Cry1a is localized to the eye in structures consistent with a role in magnetosensing (1). Furthermore, immunohistochemical staining of the avian retina showed that Cry1a undergoes conformational change in response to light at the wavelengths (UV, blue, green) which are active in mediating magnetosensitivy. Significantly, birds are able to orient to the earth’s magnetic field in near-UV and green (560nm) light in addition to blue light, and also are oriented under light pulse conditions wherein the magnetic field is applied only in the dark intervals between light pulses. These behavioural, obtained in multiple studies over 30 years from different labs, data exclude any possiblity that the avian Cry4 could function as magnetoreceptor via formation Trp/Flavin radical pairs, as has been recently suggested (2), since Cry4 does not occur in structures that can absorb UV light, and since the Trp/Flavin radical pair is not formed in green (565nm) light. This leaves Cry1a as the only possible candidate cryptochrome magnetosensor; however due to failure of prior efforts to purify rCry1a with flavin bound, the photochemical properties of the Cry1a have remained elusive. Here we provide evidence for two key properties of the avian Cry1a protein that support its role as a possible magnetosensor; namely its light wavelength sensitivity and its ability to undergo biologically relevant conformational change. The avian Cry1a was over-expressed in a recombinant mammalian HEK cell expression system. The expressing cells were passaged in the dark, then illuminated at differing wavelengths of light. Conformational change in the Cry1a protein was detected using a C-terminal antibody that specifically recognizes (binds) the light-activated form of the protein, but not the inactive state. Under UV, blue, turquoise, and green light, a significantly higher concentration of rCRY1a was immunoprecipitated with this antibody than in red light (650) or darkness. This indicated that the rC1A underwent C-terminal conformational change in response to light in this heterologous system. As a more direct probe for conformational change in rC1A, a small 13aa. epitope from the luciferase gene was introduced into various locations within the C-terminal domain of the rC1a gene, and the resulting construct expressed in HEK cells. When this epitope is exposed at the rC1A protein surface, it results in a strong bioluminescence signal (Promega inc. HIBIT lytic system). Illumination with UV, blue, or green light induced a strong bioluminescence signal change whereas red light or darkness did not. In this way, direct evidence of a light-dependent conformational change at the C-terminal of the rC1A was obtained, which moreover corresponded to the wavelength sensitivity of navigation. Finally, we discuss our results from in vivo spectroscopic analyses supporting that the Cry1a protein is flavin bound in vivo but loses the flavin upon storage, cell lysis and/or purification. In conclusion, our data support a mechanism wherein flavin in the avian Cry1a occurs as a mixture of oxidized/radical redox forms in vivo which, upon photoreduction, undergo conformational change to the biologically active reduced (FADH-) form. These findings fit both the wavelength spectrum for avian magnetosensing and the histochemical data for activation of Cry1a in the bird retina, and thereby provide additional support for Cry1a as an avian magnetosensor.