Cryptochrome 2 from Lilium × formolongi Regulates Photoperiodic Flowering in Transgenic Arabidopsis thaliana

The photoperiodic flowering pathway is essential for plant reproduction. As blue and ultraviolet-A light receptors, cryptochromes play an important role in the photoperiodic regulation of flowering. Lilium × formolongi is an important cut flower that flowers within a year after seed propagation. Floral induction is highly sensitive to photoperiod. In this study, we isolated the CRYPTOCHROME2 gene (LfCRY2) from L. × formolongi. The predicted LfCRY2 protein was highly homologous to other CRY2 proteins. The transcription of LfCRY2 was induced by blue light. LfCRY2 exhibits its highest diurnal expression during the floral induction stage under both long-day and short-day photoperiods. Overexpression of LfCRY2 in Arabidopsis thaliana promoted flowering under long days but not short days, and inhibited hypocotyl elongation under blue light. Furthermore, LfCRY2 was located in the nucleus and could interact with L. × formolongi CONSTANS-like 9 (LfCOL9) and A. thaliana CRY-interacting basic-helix-loop-helix 1 (AtCIB1) in both yeast and onion cells, which supports the hypothesis that LfCRY2 hastens the floral transition via the CIB1-CO pathway in a manner similar to AtCRY2. These results provide evidence that LfCRY2 plays a vital role in promoting flowering under long days in L. × formolongi.

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.

In addition to cryptochrome 2, magnetic particles with olfactory co-receptor are important for magnetic orientation in termites

The volatile trail pheromone is an ephemeral chemical cue, whereas the geomagnetic field (GMF) provides a stable positional reference. However, it is unclear whether and how the cryptic termites perceive the GMF for orientation in light or darkness until now. Here, we found that the two termite species, Reticulitermes chinensis and Odontotermes formosanus, use the GMF for orientation. Our silencing cryptochrome 2 (Cry2) impaired magnetic orientation in white light but had no significant impact in complete darkness, suggesting that Cry2 can mediate magnetic orientation in termites only under light. Coincidentally, the presence of magnetic particles enabled the magnetic orientation of termites in darkness. When knock-downing the olfactory co-receptor (Orco) to exclude the effect of trail pheromone, unexpectedly, we found that the Orco participated in termite magnetic orientation under both light and darkness. Our findings revealed a novel magnetoreception model depending on the joint action of radical pair, magnetic particle, and olfactory co-receptor.

Structural differences in the FAD-binding pockets and lid loops of mammalian CRY1 and CRY2 for isoform-selective regulation

The circadian clock is a biological timekeeper that operates through transcription–translation feedback loops in mammals. Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) are highly conserved core clock components having redundant and distinct functions. We recently identified the CRY1- and CRY2-selective compounds KL101 and TH301, respectively, which provide useful tools for the exploration of isoform-selective CRY regulation. However, intrinsic differences in the compound-binding FAD (flavin adenine dinucleotide) pockets between CRY1 and CRY2 are not well understood, partly because of nonoptimal properties of previously reported apo form structures in this particular region constituted by almost identical sequences. Here, we show unliganded CRY1 and CRY2 crystal structures with well-defined electron densities that are largely free of crystal contacts at the FAD pocket and nearby lid loop. We revealed conformational isomerism in key residues. In particular, CRY1 W399 and corresponding CRY2 W417 in the FAD pocket had distinct conformations (“out” for CRY1 and “in” for CRY2) by interacting with the lid loop residues CRY1 Q407 and CRY2 F424, respectively, resulting in different overall lid loop structures. Molecular dynamics simulations supported that these conformations were energetically favorable to each isoform. Isoform-selective compounds KL101 and TH301 preferred intrinsic “out” and “in” conformations of the tryptophan residue in CRY1 and CRY2, respectively, while the nonselective compound KL001 fit to both conformations. Mutations of lid loop residues designed to perturb their isoform-specific interaction with the tryptophan resulted in reversed responses of CRY1 and CRY2 to KL101 and TH301. We propose that these intrinsic structural differences of CRY1 and CRY2 can be targeted for isoform-selective regulation.

The Magnetic Compass of Birds: The Role of Cryptochrome

The geomagnetic field provides directional information for birds. The avian magnetic compass is an inclination compass that uses not the polarity of the magnetic field but the axial course of the field lines and their inclination in space. It works in a flexible functional window, and it requires short-wavelength light. These characteristics result from the underlying sensory mechanism based on radical pair processes in the eyes, with cryptochrome suggested as the receptor molecule. The chromophore of cryptochrome, flavin adenine dinucleotide (FAD), undergoes a photocycle, where radical pairs are formed during photo-reduction as well as during re-oxidation; behavioral data indicate that the latter is crucial for detecting magnetic directions. Five types of cryptochromes are found in the retina of birds: cryptochrome 1a (Cry1a), cryptochrome 1b, cryptochrome 2, cryptochrome 4a, and cryptochrome 4b. Because of its location in the outer segments of the ultraviolet cones with their clear oil droplets, Cry1a appears to be the most likely receptor molecule for magnetic compass information.

Multiple Sclerosis-Associated hnRNPA1 Mutations Alter hnRNPA1 Dynamics and Influence Stress Granule Formation

Evidence indicates that dysfunctional heterogeneous ribonucleoprotein A1 (hnRNPA1; A1) contributes to the pathogenesis of neurodegeneration in multiple sclerosis. Understanding molecular mechanisms of neurodegeneration in multiple sclerosis may result in novel therapies that attenuate neurodegeneration, thereby improving the lives of MS patients with multiple sclerosis. Using an in vitro, blue light induced, optogenetic protein expression system containing the optogene Cryptochrome 2 and a fluorescent mCherry reporter, we examined the effects of multiple sclerosis-associated somatic A1 mutations (P275S and F281L) in A1 localization, cluster kinetics and stress granule formation in real-time. We show that A1 mutations caused cytoplasmic mislocalization, and significantly altered the kinetics of A1 cluster formation/dissociation, and the quantity and size of clusters. A1 mutations also caused stress granule formation to occur more quickly and frequently in response to blue light stimulation. This study establishes a live cell optogenetics imaging system to probe localization and association characteristics of A1. It also demonstrates that somatic mutations in A1 alter its function and promote stress granule formation, which supports the hypothesis that A1 dysfunction may exacerbate neurodegeneration in multiple sclerosis.

Structural insights into photoactivation of plant Cryptochrome-2

Cryptochromes (CRYs) are evolutionarily conserved photoreceptors that mediate various light-induced responses in bacteria, plants, and animals. Plant cryptochromes govern a variety of critical growth and developmental processes including seed germination, flowering time and entrainment of the circadian clock. CRY’s photocycle involves reduction of their flavin adenine dinucleotide (FAD)-bound chromophore, which is completely oxidized in the dark and semi to fully reduced in the light signaling-active state. Despite the progress in characterizing cryptochromes, important aspects of their photochemistry, regulation, and light-induced structural changes remain to be addressed. In this study, we determine the crystal structure of the photosensory domain of Arabidopsis CRY2 in a tetrameric active state. Systematic structure-based analyses of photo-activated and inactive plant CRYs elucidate distinct structural elements and critical residues that dynamically partake in photo-induced oligomerization. Our study offers an updated model of CRYs photoactivation mechanism as well as the mode of its regulation by interacting proteins.

Arabidopsis CIB3 regulates photoperiodic flowering in an FKF1-dependent way

ABSTRACT Arabidopsis cryptochrome 2 (CRY2) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) are blue light receptors mediating light regulation of growth and development, such as photoperiodic flowering. CRY2 interacts with a basic helix-loop-helix (bHLH) transcription factor CIB1 in response to blue light to activate the transcription of the flowering integrator gene FLOWERING LOCUS T (FT). CIB1, CIB2, CIB4 and CIB5 function redundantly to promote flowering in a CRY2-dependent way and form various heterodimers to bind to the non-canonical E-box sequence in the FT promoter. However, the function of CIB3 has not been described. We discovered that CIB3 promotes photoperiodic flowering independently of CRY2. Moreover, CIB3 does not interact with CRY2 but interacts with CIB1 and functions synergistically with CIB1 to promote transcription of the GI gene. FKF1 is required for CIB3 to promote flowering and enhances the CIB1-CIB3 interaction in response to blue light.

Light-Regulated Transcription of a Mitochondrial-Targeted K+ Channel

The inner membranes of mitochondria contain several types of K+ channels, which modulate the membrane potential of the organelle and contribute in this way to cytoprotection and the regulation of cell death. To better study the causal relationship between K+ channel activity and physiological changes, we developed an optogenetic platform for a light-triggered modulation of K+ conductance in mitochondria. By using the light-sensitive interaction between cryptochrome 2 and the regulatory protein CIB1, we can trigger the transcription of a small and highly selective K+ channel, which is in mammalian cells targeted into the inner membrane of mitochondria. After exposing cells to very low intensities (≤0.16 mW/mm2) of blue light, the channel protein is detectable as an accumulation of its green fluorescent protein (GFP) tag in the mitochondria less than 1 h after stimulation. This system allows for an in vivo monitoring of crucial physiological parameters of mitochondria, showing that the presence of an active K+ channel causes a substantial depolarization compatible with the effect of an uncoupler. Elevated K+ conductance also results in a decrease in the Ca2+ concentration in the mitochondria but has no impact on apoptosis.