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Mosquito disease vector control relies mostly on toxic insecticides. A more environmentally friendly alternative is to make use of light-based behavioral manipulation to attract pests to traps and repel pests away from human habitation. The present technology is based on the assumption that mosquito UV light detection occurs solely through opsin-based photoreception in the eyes. The Holmes Lab has recently found additional UV and short wavelength photoreceptive elements expressed in central brain neurons that strongly modulate complex insect behavioral responses to light. Therefore, there is a need to incorporate these additional elements in disease vector control designs for improved efficiency. Namely, CRYPTOCHROME (CRY), which is classically associated with its role in circadian clock resetting, activates with blue- and UV- light and increases the electrical excitability of circadian/arousal neurons. I hypothesized that CRY and other photoreceptor systems mediate photic input to the circadian/arousal circuit of flies in order to mediate specific light-based behavioral responses. To test this, I modified and adapted light-evoked electrophysiology protocols to test the photoexcitability of primary arousal neurons in response to different stimuli of light using several photoreceptor mutants that lack one of three photoreceptor systems, as well as transgenic flies expressing mosquito CRY1 from a diurnal and nocturnal species. I also modified and adapted several behavioral assays to determine light attraction/avoidance as well as light-pulse arousal behavioral response to these different mutants. These modifications involved developing a new light emitting apparatus that improved on intensity control and spatiotemporal light exposure with a finer tuned spectral emission for UV, violet, blue, and red light. In this dissertation, I found that CRY coordinates with other photoreceptors to mediate light-induced electrical excitability of neurons, which underlie complex sleep/wake and light arousal behaviors. Furthermore, I found that CRY phototransduction persists across species in an intensity-dependent and species-specific manner, controlling light behavioral phototaxis across diurnal and nocturnal mosquito CRY1 profiles. I conclude with non-image forming as an amalgamation of multiple photoreceptor system contributions over a broad spectral range that affect downstream behavioral processes, including CRY as a primary short-wavelength photoreceptor for flies and mosquitos.

How cells respond to light or time is a fundamental question in biology. Cryptochromes (CRYs) are evolutionarily conserved blue light receptors or key components of the circadian oscillator found in major evolutionary lineages, from bacteria to human and have been intensively studied. However, the structure-function relationship of CRYs from evolutionary perspective is unclear. In this thesis, I interrogated the evolutionary roles of universally conserved residues (UCRs) of Arabidopsis thaliana cryptochrome 2 (CRY2) (Chapter 2) and developed optogenetic tools by engineering CRY2 using continuous directed evolution techniques (Chapter 3).UCRs are invariable amino acids evolutionarily conserved among members of a protein family across diverse kingdoms of life. UCRs are considered important for stability and/or function of protein families, but it has not been experimentally examined systematically. In Chapter 2, I experimentally analyzed 51 UCRs of Arabidopsis CRY2 that are universally conserved in eukaryotic cryptochromes from Arabidopsis to human. Surprisingly, I found that UCRs required for stable protein expression of CRY2 in plants are not similarly required for stable protein expression of human hCRY1 in human cells. Moreover, 74% of the stably expressed CRY2 proteins mutated in UCRs retained wild-type-like activities for at least one of the photoresponses I analyzed. My finding suggests that the evolutionary mechanisms underlying conservation of UCRs or that distinguish UCRs from non-UCRs determining the same functions of individual cryptochromes remain to be investigated.CRY2 mainly regulates plant photomorphogenesis through blue-light-specific interactions with numerous protein partners. Such blue-light-specific interactions have been exploited in optogenetics to manipulated biological events in a timely and precisely manner. Chapter 3 focused on the development of a novel pair of blue-light-dependent interacting proteins: CRY2-BIC1 (Blue-light Inhibitor of Cryptochromes 1) and applied PACE (Phage Assisted Continuous Evolution) to increase the dynamic range of CRY2-BIC1 blue-light dependent interaction. I isolated variants of CRY2 with stronger interactions with BIC1 and developed soluble expression and protein-dissociating PACE to facilitate further engineering of CRY2, which could give hints on characteristics of UCRs and non-UCRs.

Lateral ventral neurons (LNvs) in the fly circadian neural circuit mediate behaviors other than clock resetting, including light-activated acute arousal. Converging sensory inputs often confer functional redundancy. The LNvs have three distinct light input pathways: (1) cell autonomously expressed cryptochrome (CRY), (2) rhodopsin 7 (Rh7), and (3) synaptic inputs from the eyes and other external photoreceptors that express opsins and CRY. We explored the relative photoelectrical and behavioral input contributions of these three photoreceptor systems to determine their functional impact in flies. Patch-clamp electrophysiology measuring light evoked firing frequency (FF) was performed on large LNvs (l-LNvs) in response to UV (365 nm), violet (405 nm), blue (450 nm), or red (635 nm) LED light stimulation, testing controls versus mutants that lack photoreceptor inputs gl60j, cry-null, rh7-null, and double mutant gl60j-cry-null flies. For UV, violet, and blue short wavelength light inputs, all photoreceptor mutants show significantly attenuated action potential FF responses measured in the l-LNv. In contrast, red light FF responses are only significantly attenuated in double mutant gl60j-cry-null flies. We used a light-pulse arousal assay to compare behavioral responses to UV, violet, blue and red light of control and light input mutants, measuring the awakening arousal response of flies during subjective nighttime at two different intensities to capture potential threshold differences (10 and 400 μW/cm2). The light arousal behavioral results are similar to the electrophysiological results, showing significant attenuation of behavioral light responses for mutants compared to control. These results show that the different LNv convergent photoreceptor systems are integrated and together confer functional redundancy for light evoked behavioral arousal.

Nearly all walks of life, from single cell cyanobacteria to humans, have evolved an intimate connection to the light dark cycle and coordinate physiology and behavior to the solar day. This phenomenon is known as circadian rhythms and is an adaptation that organisms use to anticipate daily environmental changes. In mammals, disruption of circadian rhythms through environmental stimulus or genetic means leads to the onset of many diseases such as: diabetes, cardiovascular disease, premature aging and cancer. Nearly every cell in the human body has an endogenous molecular clock that controls integrated biochemical processes on a ~24-hour period. At the core of this molecular clock is the heterodimeric basic helix-loop-helix Per:Arnt:Sim (bHLH-PAS) transcription factor CLOCK:BMAL1 that together with its repressors, Period (PER) and Cryptochrome (CRY), forms an autoregulatory feedback loop that results in the rhythmic transcription of nearly 40% of the genome including essential genes in metabolism, hormone secretion and the cell cycle. While this model for transcription-translation feedback loop has been accepted for over two decades, it is still not well understood how CLOCK:BMAL1 activity is directly regulated to generate intrinsic 24-hour timing in mammals. Using a combination of biochemistry, structural and cell biology we have elucidated a mechanism by which CRY1 directly interacts with CLOCK:BMAL1 to form a critical repressive circadian complex in circadian rhythms. We have also discovered an uncharacterized PAS domain containing protein in humans, PAS Domain containing protein 1 (PASD1), that inhibits CLOCK:BMAL1 activity and suppresses circadian cycling in cancer cells, providing a molecular link from oncogenesis to circadian disruption.Chapter 2 describes our work to elucidate how CRY1 directly inhibits CLOCK:BMAL1 activity. CRYs close the tightly regulated transcriptional feedback loop to control circadian rhythms, however, the mechanistic underpinnings of how CRYs interact with and repress CLOCK:BMAL1 have remained elusive. Previous studies in our lab showed that tuning affinity of CRY1 for the transactivation domain (TAD) of BMAL1 controls circadian period by competing with the coactivator CBP/p300. CRY1 also binds to CLOCK, although it was not yet understood how multivalent interactions with CLOCK:BMAL1 contribute to CRY1 function. I have shown that CRY1 directly binds the CLOCK:BMAL1 PAS-AB core and this interaction is driven by a single domain in this multi-domain protein complex, CLOCK PAS-B. Furthermore, I have worked with experts in small angle x-ray scattering analysis to generate a model of the CRY1:CLOCK:BMAL1 complex that situates CRY1 atop the CLOCK:BMAL bHLH PAS-AB domains in the solution envelope providing the first low resolution description of the CRY1:CLOCK:BMAL1 complex. These studies have paved the way for circadian-directed therapeutics and have provided a basis for comparative analysis between the CRY1 and CRY2 proteins as described in Chapter 5. Further studies on the assembly of circadian repressive complexes, including CRY and PERIOD proteins, is described in Chapter 6. In Chapter 4 we report the discovery of a previously uncharacterized repressor of circadian rhythms, PAS domain containing protein 1 (PASD1). Upon joining Dr. Partch’s lab I chose to work on the “long-shot project” – an interesting but undeveloped project that was based entirely off Dr. Partch’s initial discovery of an uncharacterized gene that bears significant similarity to a clock protein, potentially possessing the ability to modulate the circadian clock. This CLOCK-like protein, PASD1, is not expressed in healthy somatic tissues, but is instead limited to gametogenic tissues where there are no functional clocks. Using transcriptional reporter assays I confirmed initial results that PASD1 inhibits transactivation of genes by the core circadian transcription factor, CLOCK:BMAL1. Through series of truncation experiments I found that the C-terminus of PASD1 is sufficient to repress CLOCK:BMAL1 in the nucleus. Furthermore, deletion of one region that is highly conserved with CLOCK alleviates repression by PASD1 to suggest it utilizes molecular mimicry to interfere with CLOCK:BMAL1 function. Furthermore, knockdown of PASD1 in both colon and lung cancer cell lines improves the amplitude of cycling, indicative of a more robust oscillator. These data together provide a tool to rescue the clock in PASD1+ tumors. In summary, I have used biochemistry and biophysics to describe how the essential circadian protein, CRY1, serves as a potent repressor of CLOCK:BMAL1 activity to establish circadian rhythms. This work provides molecular details of the CRY1-CLOCK:BMAL1 interaction that are being actively used to create circadian-based therapeutics and study the structure of circadian repressive complexes. I have also used cell biology to identify a novel repressor of CLOCK:BMAL1 that is highly up-regulated in many forms of cancer and suppresses circadian cycling.

Nearly all terrestrial organisms possess an intrinsic biological timekeeping system that functions to align key physiological processes with the solar day. In mammals, this circadian cycling is driven by temporally specific interactions between the heterodimeric transcription factor, CLOCK:BMAL1 and its cognate transcriptional regulators. The biochemical processes that drive the handoff between active and repressive complexes tunes the phase, period and robustness of circadian cycling. In this study, we show that the unstructured C-terminal transactivation domain (TAD) of BMAL1 is a regulatory hub where transcriptional activators and repressors compete for binding. Using NMR spectroscopy and real time monitoring of circadian cycling in mammalian cells we studied how interactions of the TAD with transcriptional regulators control the generation of circadian rhythms. We show that the KIX and TAZ1 domains of CBP and the CC helix of CRY1 interact with the BMAL1 TAD at overlapping sites. Perturbations of these regions of BMAL1 by site-directed mutagenesis have direct consequences for the balance of transcriptional activation and repression and elicit large changes the period of circadian oscillations. Using paramagnetic resonance spectroscopy we show that the BMAL1 TAD undergoes significant and differing conformational rearrangements upon binding CBP KIX and CRY CC. Furthermore, we identified a slow conformational change in the extreme C-terminus of BMAL1, a region of the TAD that is essential for normal circadian timekeeping. Using NMR spectroscopy, we performed structural and kinetic analysis of the conformational switch to identify a cis/trans isomerization about a Trp-Pro imide bond. Using NMR, fluorescence polarization and cell-based studies, we further characterized the switch to assess its role in circadian cycling and identified cyclophilins capable of regulating the rate of switch interconversion in vitro. Together, these data highlight the importance of structural dynamics of the BMAL1 TAD in tuning the molecular circadian clock.

While migratory birds have long been known to use the Earth's magnetic field for navigation, the precise biophysical mechanism behind this magnetic sense remains unconfirmed. A leading theory of magnetoreception suggests a chemical compass model with a yet undetermined molecular reaction site and unknown magnetically sensitive reactants. The cryptochrome photoreceptor has emerged as a promising candidate site. This investigation numerically models the first order kinetics of cryptochrome mediated photoreception, in order to evaluate its ability to function as a magnetic sensor and transduce orientation information along a neural pathway. A signal-to-noise ratio is defined to quantify the threshold for the functioning of a cryptochrome-based chemical compass. The model suggests that a flavin-superoxide radical pair in cryptochrome functions as the chemical reactants for magnetoreception. Such a cryptochrome-based signal transduction model reasonably predicts the general light intensity and wavelength effects that have been experimentally observed in migratory birds.

Disruption of circadian rhythms increases the risk of several types of cancer. Mammalian cryptochromes (CRY1 and CRY2) are circadian transcriptional repressors that are related to DNA repair enzymes. While CRYs lack DNA repair activity, they modulate the transcriptional response to DNA damage, and CRY2 can promote SCFFBXL3-mediated ubiquitination of c-MYC and other targets. Here, we characterize five mutations in CRY2 observed in human cancers in The Cancer Genome Atlas. We demonstrate that two orthologous mutations of mouse CRY2 (D325H and S510L) accelerate the growth of primary mouse fibroblasts expressing high levels of c-MYC. Neither mutant affects steady state levels of overexpressed c-MYC, and they have divergent impacts on circadian rhythms and on the ability of CRY2 to interact with SCFFBXL3. Unexpectedly, stable expression of either CRY2 D325H or of CRY2 S510L robustly suppresses P53 target gene expression, suggesting that this is the primary mechanism by which they influence cell growth.

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., Palayam, Ganapathy, Guercio et al. determine the crystal structure of the photosensory domain of Arabidopsis Cryptochrome-2 (CRY2) in a tetrameric active state, identifying critical residues that participate in photo-induced oligomerization of CRY2. This study offers an updated model of CRY’s photoactivation mechanism.

Cryptochrome (CRY) proteins play an essential role in regulating mammalian circadian rhythms. CRY is composed of a structured N-terminal domain known as the photolyase homology region (PHR), which is tethered to an intrinsically disordered C-terminal tail. The PHR domain is a critical hub for binding other circadian clock components such as CLOCK, BMAL1, PERIOD, or the ubiquitin ligases FBXL3 and FBXL21. While the isolated PHR domain is necessary and sufficient to generate circadian rhythms, removing or modifying the cryptochrome tails modulates the amplitude and/or periodicity of circadian rhythms, suggesting that they play important regulatory roles in the molecular circadian clock. In this commentary, we will discuss how recent studies of these intrinsically disordered tails are helping to establish a general and evolutionarily conserved model for CRY function, where the function of PHR domains is modulated by reversible interactions with their intrinsically disordered tails. Video abstract Supplementary information Supplementary information accompanies this paper at 10.1186/s12964-020-00665-z.

Circadian rhythms are generated by interlocked transcription-translation feedback loops that establish cell-autonomous biological timing of ∼24 h. Mutations in core clock genes that alter their stability or affinity for one another lead to changes in circadian period. The human CRY1Δ11 mutant lengthens circadian period to cause delayed sleep phase disorder (DSPD), characterized by a very late onset of sleep. CRY1 is a repressor that binds to the transcription factor CLOCK:BMAL1 to inhibit its activity and close the core feedback loop. We previously showed how the PHR (photolyase homology region) domain of CRY1 interacts with distinct sites on CLOCK and BMAL1 to sequester the transactivation domain from coactivators. However, the Δ11 variant alters an intrinsically disordered tail in CRY1 downstream of the PHR. We show here that the CRY1 tail, and in particular the region encoded by exon 11, modulates the affinity of the PHR domain for CLOCK:BMAL1. The PHR-binding epitope in exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and the subunit CLOCK. Moreover, PHR-tail interactions are conserved in the paralog CRY2 and reduced when either CRY is bound to the circadian corepressor PERIOD2. Discovery of this autoregulatory role for the mammalian CRY1 tail and conservation of PHR-tail interactions in both mammalian cryptochromes highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity.

How birds sense the variations in Earth’s magnetic field for navigation is poorly understood, although cryptochromes, proteins homologous to photolyases, have been proposed to participate in this magnetic sensing. Here, in electrochemical studies with an applied magnetic field, we monitor the repair of cyclobutane pyrimidine dimer lesions in duplex DNA by photolyase, mutants of photolyase, and a modified cryptochrome. We find that the yield of dimer repair is dependent on the strength and angle of the applied magnetic field even when using magnetic fields weaker than 1 gauss. This high sensitivity to weak magnetic fields depends upon a fast radical pair reaction on the thymines leading to repair. These data illustrate chemically how cyclobutane pyrimidine dimer repair may be used in a biological compass informed by variations in Earth’s magnetic field., DNA electrochemistry studies illustrate how thymine dimer repair by photolyase and a truncated cryptochrome depends upon weak magnetic fields and thus may serve as a biological compass.

Many insects show strong behavioral responses to short wavelength light. Drosophila melanogaster exhibit Cryptochrome- and Hyperkinetic-dependent blue and ultraviolet (UV) light avoidance responses that vary by time-of-day, suggesting that these key sensory behaviors are circadian regulated. Here we show mutant flies lacking core clock genes exhibit defects in both time-of-day responses and valence of UV light avoidance/attraction behavior. Non-genetic environmental disruption of the circadian clock by constant UV light exposure leads to complete loss of rhythmic UV light avoidance/attraction behavior. Flies with ablated or electrically silenced circadian lateral ventral neurons have attenuated avoidance response to UV light. We conclude that circadian clock proteins and the circadian lateral ventral neurons regulate both the timing and the valence of UV light avoidance/attraction. These results provide mechanistic support for Pittendrigh's "escape from light" hypothesis regarding the co-evolution of phototransduction and circadian systems.

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 pathway may 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.

Significance Many animals exhibit behavioral responses to UV light, including harmful insects. Recently, the explosive spread of diseases carried by mosquitoes has increased motivation to better understand insect UV phototransduction. CRYPTOCHROME (CRY) is a highly conserved nonopsin photoreceptor expressed in a small number of brain circadian and arousal neurons in Drosophila melanogaster that mediates cell-autonomous electrophysiological membrane excitability in response to UV light. CRY signaling modulates multiple fly behaviors evoked by UV light, including acute nighttime arousal responses to light flashes and phototaxis toward low-intensity UV light. Loss of CRY or the redox sensor HYPERKINETIC (HK) leads to the loss of ability to avoid high-intensity UV light; thus, CRY signaling exhibits novel features of behavioral executive choice.

In nature, plants integrate a wide range of light signals from solar radiation to adapt to the surrounding light environment, and these light signals also regulate a variety of important agronomic traits. Blue light-sensing cryptochrome (cry) and red/far-red light-sensing phytochrome (phy) play critical roles in regulating light-mediated physiological responses via the regulated transcriptional network. Accumulating evidence in the model plant Arabidopsis has revealed that crys and phys share two mechanistically distinct pathways to coordinately regulate transcriptional changes in response to light. First, crys and phys promote the accumulation of transcription factors that regulate photomorphogenesis, such as HY5 and HFR1, via the inactivation of the CONSTITUTIVE PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 E3 ligase complex by light-dependent binding. Second, photoactive crys and phys directly interact with PHYTOCHROME INTERACTING FACTOR transcription factor family proteins to regulate transcriptional activity. The coordinated regulation of these two pathways (and others) by crys and phys allow plants to respond with plasticity to fluctuating light environments in nature.

Cryptochromes are evolutionarily related to the light-dependent DNA repair enzyme photolyase, serving as major regulators of circadian rhythms in insects and vertebrate animals. There are two types of cryptochromes in the animal kingdom: Drosophila-like CRYs that act as non-visual photopigments linking circadian rhythms to the environmental light/dark cycle, and vertebrate-like CRYs that do not appear to sense light directly, but control the generation of circadian rhythms by acting as transcriptional repressors. Some animals have both types of CRYs, while others possess only one. Cryptochromes have two domains, the photolyase homology region (PHR) and an extended, intrinsically disordered C-terminus. While all animal CRYs share a high degree of sequence and structural homology in their PHR domains, the C-termini are divergent in both length and sequence identity. Recently, cryptochrome function has been shown to extend beyond its pivotal role in circadian clocks, participating in regulation of the DNA damage response, cancer progression, and glucocorticoid signaling, as well as being implicated as possible magnetoreceptors. In this review, we provide a historical perspective on the discovery of animal cryptochromes, examine similarities and differences of the two types of animal cryptochromes, and explore some of the divergent roles for this class of proteins.

International audience; Cryptochromes are flavoprotein photoreceptors with multiple signaling roles during plant de-etiolation and development. Arabidopsis cryptochromes (cry1 and cry2) absorb light through an oxidized flavin (FAD(OX)) cofactor which undergoes reduction to both FADH degrees and FADH(-) redox states. Since the FADH degrees redox state has been linked to biological activity, it is important to estimate its concentration formed upon illumination in vivo. Here we model the photocycle of isolated cry1 and cry2 proteins with a three-state kinetic model. Our model fits the experimental data for flavin photoconversion in vitro for both cry1 and cry2, providing calculated quantum yields which are significantly lower in cry1 than for cry2. The model was applied to the cryptochrome photocycle in vivo using biological activity in plants as a readout for FADH concentration. The fit to the in vivo data provided quantum yields for cry1 and cry2 flavin reduction similar to those obtained in vitro, with decreased cry1 quantum yield as compared to cry2. These results validate our assumption that FADH concentration correlates with biological activity. This is the first reported attempt at kinetic modeling of the cryptochrome photocycle in relation to macroscopic signaling events in vivo, and thereby provides a theoretical framework to the components of the photocycle that are necessary for cryptochrome response to environmental signals.

Cryptochromes are photolyase-like blue light receptors that are conserved in plants and animals. Although the light-dependent catalytic mechanism of photolyase is well studied, the photochemical mechanism of cryptochromes remains largely unknown. Lack of an appropriate protein expression system to obtain photochemically active cryptochrome holoproteins is a technical obstacle for the study of plant cryptochromes. We report here an easy-to-use method to express and study Arabidopsis cryptochrome in HEK293T cells. Our results indicate that Arabidopsis cryptochromes expressed in HEK293T are photochemically active. We envision a broad use of this method in the functional investigation of plant proteins, especially in the large-scale analyses of photochemical activities of cryptochromes such as blue light-dependent protein-protein interactions.

© 2015, National Academy of Sciences. All rights reserved. Cryptochromes in different evolutionary lineages act as either photoreceptors or light-independent transcription repressors. The flavin cofactor of both types of cryptochromes can be photo-reduced in vitro by electron transportation via three evolutionarily conserved tryptophan residues known as the "Trp triad." It was hypothesized that Trp triad-dependent photoreduction leads directly to photoexcitation of cryptochrome photoreceptors. We tested this hypothesis by analyzing mutations of Arabidopsis cryptochrome 1 (CRY1) altered in each of the three Trp-triad tryptophan residues (W324, W377, and W400). Surprisingly, in contrast to a previous report all photoreduction-deficient Trp-triad mutations of CRY1 remained physiologically and biochemically active in Arabidopsis plants. ATP did not enhance rapid photoreduction of the wild-type CRY1, nor did it rescue the defective photoreduction of the CRY1W324Aand CRY1W400Fmutants that are photophysiologically active in vivo. The lack of correlation between rapid flavin photoreduction or the effect of ATP on the rapid flavin photoreduction and the in vivo photophysiological activities of plant cryptochromes argues that the Trp triad-dependent photoreduction is not required for the function of cryptochromes and that further efforts are needed to elucidate the photoexcitation mechanism of cryptochrome photoreceptors.

Blue light activation of the photoreceptor CRYPTOCHROME (CRY) evokes rapid depolarization and increased action potential firing in a subset of circadian and arousal neurons in Drosophila melanogaster. Here we show that acute arousal behavioral responses to blue light significantly differ in mutants lacking CRY, as well as mutants with disrupted opsin-based phototransduction. Light-activated CRY couples to membrane depolarization via a well conserved redox sensor of the voltage-gated potassium (K(+)) channel β-subunit (Kvβ) Hyperkinetic (Hk). The neuronal light response is almost completely absent in hk(-/-) mutants, but is functionally rescued by genetically targeted neuronal expression of WT Hk, but not by Hk point mutations that disable Hk redox sensor function. Multiple K(+) channel α-subunits that coassemble with Hk, including Shaker, Ether-a-go-go, and Ether-a-go-go-related gene, are ion conducting channels for CRY/Hk-coupled light response. Light activation of CRY is transduced to membrane depolarization, increased firing rate, and acute behavioral responses by the Kvβ subunit redox sensor.

International audience; Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH ? radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pK a of aspartic acid D396, the putative proton donor to FAD? 2 , from ,7.4 to .9, and favours a reaction pathway yielding long-lived aspartate D396 2. Its negative charge could trigger conformational changes necessary for signal transduction.

Cryptochromes are blue light receptors that regulate light responses in plants, including various crops. The molecularmechanism of plant cryptochromes has been extensively investigated in Arabidopsis thaliana, but it has not been reported in any crop species. Here, we report a study of the mechanism of soybean (Glycine max) cryptochrome2 (CRY2a). We found that CRY2a regulates leaf senescence, which is a life history trait regulated by light and photoperiods via previously unknown mechanisms. We show that CRY2a undergoes blue light-dependent interaction with the soybean basic helix-loop-helix transcription activator CIB1 (for cryptochrome-interacting bHLH1) that specifically interacts with the E-box (CANNTG) DNA sequences. Analyses of transgenic soybean plants expressing an elevated or reduced level of the CRY2a or CIB1 demonstrate that CIB1 promotes leaf senescence, whereas CRY2a suppresses leaf senescence. Results of the gene expression and molecular interaction analyses support the hypothesis that CIB1 activates transcription of senescence-associated genes, such as WRKY DNA BINDING PROTEIN53b (WRKY53b), and leaf senescence. CIB1 interacts with the E-box-containing promoter sequences of the WRKY53b chromatin, whereas photoexcited CRY2a interacts with CIB1 to inhibit its DNA binding activity. These findings argue that CIBdependent transcriptional regulation is an evolutionarily conserved CRY-signaling mechanism in plants, and this mechanism is opted in evolution to mediate light regulation of different aspects of plant development in different plant species. © 2013 American Society of Plant Biologists. All rights reserved.

Plants possess multiple photoreceptors to mediate light regulation of growth and development, but it is not well understood how different photoreceptors coordinate their actions to jointly regulate developmental responses, such as flowering time. In Arabidopsis, the photoexcited cryptochrome 2 interacts with the transcription factor CRYPTOCHROME-INTERACTING basic helix-loop-helix 1 (CIB1) to activate transcription and floral initiation. We show that the CIB1 protein expression is regulated by blue light; CIB1 is highly expressed in plants exposed to blue light, but levels of the CIB1 protein decreases in the absence of blue light. We demonstrate that CIB1 is degraded by the 26S proteasome and that blue light suppresses CIB1 degradation. Surprisingly, although cryptochrome 2 physically interacts with CIB1 in response to blue light, it is not the photoreceptor mediating blue-light suppression of CIB1 degradation. Instead, two of the three light-oxygen-voltage (LOV)-domain photoreceptors, ZEITLUPE and LOV KELCH PROTEIN 2, but not FLAVIN-BINDING KELCH REPEAT 1, are required for the function and blue-light suppression of degradation of CIB1. These results support the hypothesis that the evolutionarily unrelated blue-light receptors, cryptochrome and LOV-domain F-box proteins, mediate blue-light regulation of the same transcription factor by distinct mechanisms.

In mammals, the suprachiasmatic nucleus (SCN) is the central pacemaker organizing circadian rhythms of behavior and physiology. At the cellular level, the mammalian clock consists of autoregulatory feedback loops involving a set of “clock genes,” including the Cryptochrome ( Cry) genes, Cry1 and Cry2. Experimental evidence suggests that Cry1 and Cry2 play distinct roles in circadian clock function. In mice, Cry1 is required for sustained circadian rhythms in dissociated SCN neurons or fibroblasts but not in organotypic SCN slices or at the behavioral level, whereas Cry2 is not required at any of these levels. It has been argued that coupling among SCN cellular oscillators compensates for clock gene defects to preserve oscillatory function. Here we test this hypothesis in Cry1−/− mice by first disrupting intercellular coupling in vivo using constant light (resulting in behavioral arrhythmicity) and then examining circadian clock gene expression in SCN slices at the single cell level. In this manner, we were able to test the role of intercellular coupling without drugs and while preserving tissue organization, avoiding the confounding influences of more invasive manipulations. Cry1−/− mice (as well as control Cry2−/− mice) bearing the PER2::LUC knock-in reporter were transferred from a standard light:dark cycle to constant bright light (~650 lux) to induce arrhythmic locomotor patterns. In SCN slices from these animals, we used bioluminescence imaging to monitor PER2::LUC expression in single cells. We show that SCN slices from rhythmic Cry1−/− and Cry2−/− mice had similarly high percentages of functional single-cell oscillators. In contrast, SCN slices from arrhythmic Cry1−/− mice had significantly fewer rhythmic cells than SCN slices from arrhythmic Cry2−/− mice. Thus, constant light in vivo disrupted intercellular SCN coupling to reveal a cell-autonomous circadian defect in Cry1−/− cells that is normally compensated by intercellular coupling in vivo.

The mammalian circadian clockwork is composed of a core PER/CRY feedback loop and additional interlocking loops. In particular, the ROR/REV/Bmal1 loop, consisting of ROR activators and REV-ERB repressors that regulate Bmal1 expression, is thought to “stabilize” core clock function. However, due to functional redundancy and pleiotropic effects of gene deletions, the role of the ROR/REV/Bmal1 loop has not been accurately defined. In this study, we examined cell-autonomous circadian oscillations using combined gene knockout and RNA interference and demonstrated that REV-ERBα and β are functionally redundant and are required for rhythmic Bmal1 expression. In contrast, the RORs contribute to Bmal1 amplitude but are dispensable for Bmal1 rhythm. We provide direct in vivo genetic evidence that the REV-ERBs also participate in combinatorial regulation of Cry1 and Rorc expression, leading to their phase-delay relative to Rev-erbα. Thus, the REV-ERBs play a more prominent role than the RORs in the basic clock mechanism. The cellular genetic approach permitted testing of the robustness of the intracellular core clock function. We showed that cells deficient in both REV-ERBα and β function, or those expressing constitutive BMAL1, were still able to generate and maintain normal Per2 rhythmicity. Our findings thus underscore the resilience of the intracellular clock mechanism and provide important insights into the transcriptional topologies underlying the circadian clock. Since REV-ERB function and Bmal1 mRNA/protein cycling are not necessary for basic clock function, we propose that the major role of the ROR/REV/Bmal1 loop and its constituents is to control rhythmic transcription of clock output genes., Author Summary Circadian clocks in plants, fungi, insects, and mammals all share a common transcriptional network architecture. At the cellular level, the mammalian clockwork consists of a core Per/Cry negative feedback loop and additional interlocking loops. We wished to address experimentally the contribution of the interlocking Bmal1 loop to clock function in mammals. Because behavioral rhythms do not always reflect cell-autonomous phenotypes and are subject to pleiotropic effects, we employed cell-based genetic approaches and monitored rhythms longitudinally using bioluminescent reporters of clock gene expression. We showed that REV-ERB repressors play a more prominent role than ROR activators in regulating the Bmal1 rhythm. However, significant rhythmicity remains even with constitutive expression of Bmal1, pointing to the resilience of the core loop to perturbations of the Bmal1 loop. We conclude that while the interlocking loop contributes to fine-tuning of the core loop, its primary function is to provide discrete waveforms of clock gene expression for control of local physiology. This study has important general implications not only for circadian biology across species, but also for the emerging field of systems biology that seeks to understand complex interactions in genetic networks.