Comparative properties and functions of type 2 and type 4 pigeon cryptochromes

Abstract

Two types of vertebrate cryptochromes (Crys) are currently recognized. Type 2 Crys function in the molecular circadian clock as light-independent transcriptional repressors. Type 4 Crys are a newly discovered group with unknown function, although they are flavoproteins and therefore may function as photoreceptors. It has been postulated that Crys function in light-dependent magnetoreception, which is thought to contribute towards homing and migratory behaviors. Here we have cloned and annotated the full-length pigeon ClCry1, ClCry2, and ClCry4 genes, and characterized the full-length proteins and several site-directed mutants to investigate the roles of these proteins. ClCry1 and ClCry2 are phylogenetically grouped as Type 2 Crys and thus are expected to be core components of the pigeon circadian clock. Interestingly, we find that ClCry4 is properly annotated as a Type 4 Cry. It appears that many birds possess a Type 4 Cry which, as in pigeon, is misannotated. Like the Type 2 Crys, ClCry4 is widespread in pigeon tissues. However, unlike the Type 2 Crys, ClCry4 is cytosolic, and purified ClCry4 possesses FAD cofactor, which confers characteristic UV-visible spectra as well as two photochemical activities. We find that ClCry4 undergoes light-dependent conformational change, which is a property of insect Type1 Crys involved in the insect-specific pathway of photoentrainment of the biological clock. ClCry4 can also be photochemically reduced by a mechanism common to all FAD-containing Cry family members, and this mechanism is postulated to be influenced by the geomagnetic field. Thus pigeon Crys control circadian behavior and may also have photosensory function.

Light entrainment of retinal biorhythms: cryptochrome 2 as candidate photoreceptor in mammals

“…In mice, the ability of CRY 1 to form the heterodimer CRY1/PER2 that represses the CLOCK/BMAL1-dependent transcription has been shown not to depend on FAD, in contrast to that of Drosophila CRY [25]. In line with that in vitro observation, the FAD binding pocket of vertebrate CRY 1 is unoccupied when it is either in its ground state or in its active, heterodimeric, form [32,43].…”

“…A recent study challenged these conclusions, demonstrating the lack of specificity in two commercial CRY 2 antibodies using tissue from CRY 2 knockout mice as negative controls [30]. Based on the known light dependence of the circadian function of CRY in plants and the high structural homology between type II CRY of vertebrates and plant CRY, including the Trp triad [32], it has been proposed that CRY of the retina are responsible for circadian photoreception in vertebrates [33]. To investigate the role of CRY in nonvisual photoreception in mice, Van Gelder et al [34] studied the pupillary constriction reflex in response to blue light (470 nm).…”

Cryptochromes Mediate Intrinsic Photomechanical Transduction in Avian Iris and Somatic Striated Muscle

“…myotubes with little or no CRY localization detectable in their nuclei (Figures 4B1-4). Because cytosolic CRYs have been linked to light sensitivity in avians (Mouritsen et al, 2004;Wang et al, 2018) it was expected that pectoral myotubes would also exhibit intrinsic photosensitivity. In accord with this expectation, 435/535 nm dual excitation light evoked PMT in pectoral myotubes (Figures 4C,D) featuring peak increases in Ca 2+ F/F B that were even larger than those from iris myotubes (140 ± 14% versus 49 ± 9%, respectively, p < 0.05) while displaying a similar degree and incidence of contractions (11 ± 2% shortening, in 20 of 74 myotubes).…”

“…Semi-quantitative assessment of CRY relative to GAPDH PCR amplification products revealed no effect on CRY transcripts with four of the six ASOs tested (data not shown) while two, CRY1 ASO 1.3 and CRY2 ASO 2.3, dramatically reduced CRY1 and CRY2 transcripts by 87 ± 6% (N = 4 experiments) and 91 ± 5% (N = 3 experiments) respectively, compared to CRY transcript levels in untreated controls (Figures 5A,B). Since avian CRY4 binds FAD, is expressed in striated muscle, and plays a lightdependent role in magnetoreception (Kubo et al, 2006;Watari et al, 2012;Wang et al, 2018) the possibility that CRY1 ASO 1.3 or CRY2 ASO 2.3 might knock down CRY4 transcripts and thereby reduce PMTRs was tested by amplifying CRY4 from cDNA templates derived from the same control and ASO treated cultures. This possibility seemed unlikely because Gallus mRNAs encoding CRYs 1 and 2 respectively have only 39 and 36% identity with CRY4 mRNA, and because neither CRY1 ASO 1.3 nor CRY2 ASO 2.3 is predicted to bind to corresponding sites on CRY4.…”

“…Avian CRYs pose an exception to the simple invertebrate lightdependent (Type I) versus vertebrate light-independent (Type II) classification since an avian CRY (CRY4) binds FAD and plays a light-dependent role in magnetoreception (Kubo et al, 2006;Watari et al, 2012;Wang et al, 2018). Moreover, avian CRY1 and CRY2 share 14 of the 17 amino acids with CRY4 at sites implicated in FAD binding as well as a triad of tryptophans believed to facilitate light-activated intramolecular electron transfer (Kubo et al, 2006).…”