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The circadian system regulates numerous physiological processes including immune responses. Here, we show that mice deficient of the circadian clock genes Cry1 and Cry2 [Cry double knockout (DKO)] develop an autoimmune phenotype including high serum IgG concentrations, serum antinuclear antibodies, and precipitation of IgG, IgM, and complement 3 in glomeruli and massive infiltration of leukocytes into the lungs and kidneys. Flow cytometry of lymphoid organs revealed decreased pre-B cell numbers and a higher percentage of mature recirculating B cells in the bone marrow, as well as increased numbers of B2 B cells in the peritoneal cavity of Cry DKO mice. The B cell receptor (BCR) proximal signaling pathway plays a critical role in autoimmunity regulation. Activation of Cry DKO splenic B cells elicited markedly enhanced tyrosine phosphorylation of cellular proteins compared with cells from control mice, suggesting that overactivation of the BCR-signaling pathwaymay contribute to the autoimmunity phenotype in the Cry DKO mice. In addition, the expression of C1q, the deficiency of which contributes to the pathogenesis of systemic lupus erythematosus, was significantly down-regulated in Cry DKO B cells. Our results suggest that B cell development, the BCR-signaling pathway, and C1q expression are regulated by circadian clock CRY proteins and that their dysregulation through loss of CRY contributes to autoimmunity.

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

Cryptochromes (CRYs) are a group of evolutionarily conserved flavoproteins found in many organisms. In plants, the well-studied CRY photoreceptor, activated by blue light, plays essential roles in plant growth and development. However, the mechanism of activation remains largely unknown. Here, we determined the oligomeric structures of the blue-light-perceiving PHR domain of Zea mays CRY1 and an Arabidopsis CRY2 constitutively active mutant. The structures form dimers and tetramers whose functional importance is examined in vitro and in vivo with Arabidopsis CRY2. Structure-based analysis suggests that blue light may be perceived by CRY to cause conformational changes, whose precise nature remains to be determined, leading to oligomerization that is essential for downstream signaling. This photoactivation mechanism may be widely used by plant CRYs. Our study reveals a molecular mechanism of plant CRY activation and also paves the way for design of CRY as a more efficient optical switch.Structural determination and analysis of the PHR domain of plant CRY proteins suggest that blue-light perception causes the CRY oligomerization required for downstream signaling.

•We explain mechanisms of light-induced conformational change of photoactivatable proteins.•We describe strategies and studies of using photoactivatable proteins to control intracellular signaling pathways.•We highlight the advantages of using light to control intracellular signaling pathways with superior spatial and temporal resolution.•We discuss precautions to be used in designing experimental schemes of optogenetic control of cell signaling. Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, although useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways and discuss future prospects for the field, including integration of new genetic approaches into optogenetics.

The mammalian circadian clock consists of a transcription–translation feedback loop (TTFL) composed of CLOCK–BMAL1 transcriptional activators and CRY–PER transcriptional repressors. Previous work showed that CRY inhibits CLOCK–BMAL1-activated transcription by a “blocking”-type mechanism and that CRY–PER inhibits CLOCK–BMAL1 by a “displacement”-type mechanism. While the mechanism of CRY-mediated repression was explained by both in vitro and in vivo experiments, the CRY–PER-mediated repression in vivo seemed in conflict with the in vitro data demonstrating PER removes CRY from the CLOCK–BMAL1–E-box complex. Here, we show that CRY–PER participates in the displacement-type repression by recruiting CK1δ to the nucleus and mediating an increased local concentration of CK1δ at CLOCK–BMAL1-bound promoters/enhancers and thus promoting the phosphorylation of CLOCK and dissociation of CLOCK–BMAL1 along with CRY from the E-box. Our findings bring clarity to the role of PER in the dynamic nature of the repressive phase of the TTFL.

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–Vis spectra as well as two photochemical activities. We find that ClCry4 undergoes light-dependent conformational change, which is a property of insect Type 1 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.

Photolyases and cryptochromes are evolutionarily related flavoproteins, which however perform distinct physiological functions. Photolyases (PHR) are evolutionarily ancient enzymes. They are activated by light and repair DNA damage caused by UV radiation. Although cryptochromes share structural similarity with DNA photolyases, they lack DNA repair activity. Cryptochrome (CRY) is one of the key elements of the circadian system in animals. In plants, CRY acts as a blue light receptor to entrain circadian rhythms, and mediates a variety of light responses, such as the regulation of flowering and seedling growth. We performed a comprehensive evolutionary analysis of the CRY/PHR superfamily. The superfamily consists of 7 major subfamilies: CPD class I and CPD class II photolyases, (6-4) photolyases, CRY-DASH, plant PHR2, plant CRY and animal CRY. Although the whole superfamily evolved primarily under strong purifying selection (average ω = 0.0168), some subfamilies did experience strong episodic positive selection during their evolution. Photolyases were lost in higher animals that suggests natural selection apparently became weaker in the late stage of evolutionary history. The evolutionary time estimates suggested that plant and animal CRYs evolved in the Neoproterozoic Era (~1000-541 Mya), which might be a result of adaptation to the major climate and global light regime changes occurred in that period of the Earth's geological history.

The cryptochrome/photolyase (CRY/PL) family represents an ancient group of proteins fulfilling two fundamental functions. While photolyases repair UV-induced DNA damages, cryptochromes mainly influence the circadian clock. In this study, we took advantage of the large number of already sequenced and annotated genes available in databases and systematically searched for the protein sequences of CRY/PL family members in all taxonomic groups primarily focusing on metazoans and limiting the number of species per taxonomic order to five. Using BLASTP searches and subsequent phylogenetic tree and motif analyses, we identified five distinct photolyases (CPDI, CPDII, CPDIII, 6-4 photolyase, and the plant photolyase PPL) and six cryptochrome subfamilies (DASH-CRY, mammalian-type MCRY, Drosophila-type DCRY, cnidarian-specific ACRY, plant-specific PCRY, and the putative magnetoreceptor CRY4. Manually assigning the CRY/PL subfamilies to the species studied, we have noted that over evolutionary history, an initial increase of various CRY/PL subfamilies was followed by a decrease and specialization. Thus, in more primitive organisms (e.g., bacteria, archaea, simple eukaryotes, and in basal metazoans), we find relatively few CRY/PL members. As species become more evolved (e.g., cnidarians, mollusks, echinoderms, etc.), the CRY/PL repertoire also increases, whereas it appears to decrease again in more recent organisms (humans, fruit flies, etc.). Moreover, our study indicates that all cryptochromes, although largely active in the circadian clock, arose independently from different photolyases, explaining their different modes of action.

Cryptochromes (CRY) are flavoproteins that direct a diverse array of developmental processes in response to blue light in plants. Conformational changes in CRY are induced by the absorption of photons and result in the propagation of light signals to downstream components. In Arabidopsis, CRY1 and CRY2 serve both distinct and partially overlapping functions in regulating photomorphogenic responses and photoperiodic flowering. For example, both CRY1 and CRY2 regulate the abundance of transcription factors by directly reversing the activity of E3 ubiquitin ligase on CONSTITUTIVE PHOTOMORPHOGENIC 1 and SUPPRESSOR OF PHYA-105 1 complexes in a blue light-dependent manner. CRY2 also specifically governs a photoperiodic flowering mechanism by directly interacting with a transcription factor called CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX. Recently, structure/function analysis of CRY1 revealed that the CONSTITUTIVE PHOTOMORPHOGENIC 1 independent pathway is also involved in CRY1-mediated inhibition of hypocotyl elongation. CRY1 and CRY2 thus not only share a common pathway but also relay light signals through distinct pathways, which may lead to altered developmental programs in plants.

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4 COP1/SPAs , has been reported for plant CRYs so far. Here we show that Cul3 LRBs is the second E3 ligase of CRY2 in Arabidopsis . We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4 COP1/SPAs and Cul3 LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature. The fate of proteins in cells is determined by not only synthesis but also degradation. Here Chen et al. show that degradation of the plant blue light receptor CRY2 is determined by two distinct E3 ubiquitin ligases, Cul4 COP1/SPAs and Cul3 LRBs , regulating the function of CRY2 under different light conditions.