Animal Cryptochromes Research Articles

The Cryptochromes: Blue Light Photoreceptors in Plants and Animals

Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.

Light-activated Cryptochrome Reacts with Molecular Oxygen to Form a Flavin–Superoxide Radical Pair Consistent with Magnetoreception

Cryptochromes are flavin-based photoreceptors occurring throughout the biological kingdom, which regulate growth and development in plants and are involved in the entrainment of circadian rhythms of both plants and animals. A number of recent theoretical works suggest that cryptochromes might also be the receptors responsible for the sensing of the magnetic field of the earth (e.g. in insects, migratory birds, or migratory fish). Cryptochromes undergo forward light-induced reactions involving electron transfer to excited state flavin to generate radical intermediates, which correlate with biological activity. Here, we give evidence of a mechanism for the reverse reaction, namely dark reoxidation of protein-bound flavin in Arabidopsis thaliana cryptochrome (AtCRY1) by molecular oxygen that involves formation of a spin-correlated FADH(•)-superoxide radical pair. Formation of analogous radical pairs in animal cryptochromes might enable them to function as magnetoreceptors.

Magnetoreception

Few subjects in animal behavior have more exotic mystery than magnetic-field sensitivity. A force we cannot sense, generated by events no one completely understands, creates field lines that pass through our bodies without any evident effect on us or on them. It is an energy felt as much by migrating lobsters on the sea floor as by ocean-crossing birds thousands of meters overhead, transduced in generally poorly understood ways. Despite the blindness of humans, modern life depends on this invisible, ghostlike field. Aside from lights and heaters, nearly every electrical device we own makes use of electromagnetism, and that same magnetism is essential in generating the power these new-found necessities consume. But for many animals, the reliance is far older and more basic: their life-or-death ability to find their way around in the world depends on correctly interpreting the earth's magnetic field.

Animal cryptochromes mediate magnetoreception by an unconventional photochemical mechanism.

Understanding the biophysical basis of animal magnetoreception has been one of the greatest challenges in sensory biology. Recently it was discovered that the light-dependent magnetic sense of Drosophila melanogaster is mediated by the ultraviolet (UV)-A/blue light photoreceptor cryptochrome (Cry). Here we show, using a transgenic approach, that the photoreceptive, Drosophila-like type 1 Cry and the transcriptionally repressive, vertebrate-like type 2 Cry of the monarch butterfly (Danaus plexippus) can both function in the magnetoreception system of Drosophila and require UV-A/blue light (wavelength below 420 nm) to do so. The lack of magnetic responses for both Cry types at wavelengths above 420 nm does not fit the widely held view that tryptophan triad-generated radical pairs mediate the ability of Cry to sense a magnetic field. We bolster this assessment by using a mutant form of Drosophila and monarch type 1 Cry and confirm that the tryptophan triad pathway is not crucial in magnetic transduction. Together, these results suggest that animal Crys mediate light-dependent magnetoreception through an unconventional photochemical mechanism. This work emphasizes the utility of Drosophila transgenesis for elucidating the precise mechanisms of Cry-mediated magnetosensitivity in insects and also in vertebrates such as migrating birds.

Microsecond Light-Induced Proton Transfer to Flavin in the Blue Light Sensor Plant Cryptochrome

Plant cryptochromes are blue light photoreceptors that regulate key responses in growth and daily rhythm of plants and might be involved in magnetoreception. They show structural homology to the DNA repair enzyme photolyase and bind flavin adenine dinucleotide as chromophore. Blue light absorption initiates the photoreduction from the oxidized dark state of flavin to the flavin neutral radical, which is the signaling state of the sensor. Previous time-resolved studies of the photoreduction process have been limited to observation of the decay of the radical in the millisecond time domain. We monitored faster, light-induced changes in absorption of an algal cryptochrome covering a spectral range of 375-750 nm with a streak camera setup. Electron transfer from tryptophan to flavin is completed before 100 ns under formation of the flavin anion radical. Proton transfer takes place with a time constant of 1.7 micros leading to the flavin neutral radical. Finally, the flavin radical and a tryptophan neutral radical decay with a time constant >200 micros in the millisecond and second time domain. The microsecond proton transfer has not been observed in animal cryptochromes from insects or photolyases. Furthermore, the strict separation in time of electron and proton transfer is novel in the field of flavin-containing photoreceptors. The reaction rate implies that the proton donor is not in hydrogen bonding distance to the flavin N5. Potential candidates for the proton donor and the involvement of the tryptophan triad are discussed.

Comparative Photochemistry of Animal Type 1 and Type 4 Cryptochromes

Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs. In addition, we analyzed photoinduced proteolytic degradation of both types of CRYs in vivo in heterologous systems. We find that even though both types of CRYs contain stoichiometric flavin, type 1 CRY is proteolytically degraded by a light-initiated reaction in Drosophila S2, zebrafish Z3, and human HEK293T cell lines, but zebrafish CRY4 (type 4) is not. In vivo degradation of type 1 CRYs does not require continuous illumination, and a single light flash of 1 ms duration leads to degradation of about 80% of Drosophila CRY in 60 min. Finally, we demonstrate that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs have autokinase activities.

XIX meeting of the Italian Society of Pure and Applied Biophysics

This special issue of the European Biophysics Journal contains papers presented as invited lectures or oral contributions at the XIX meeting of the Italian Society of Pure and Applied Biophysics, also known as SIBPA. The conference was held in Rome, 17–20 September 2008 at the “Sacro Cuore” Catholic University. For the second time, the Wrst was in 2006, the articles presented at the conference appear in this journal that is the major point of reference for the European Biophysical Societies’ Association (EBSA) and once again witness of the interesting development of the Italian Biophysics. The main topics of the meeting were organized in seven sessions, with two invited lectures per session, and a special lecture concerning the renewable energies obtained from biological sources performed by J. Barber of the Wolfson Labs (Imperial College, London). During the Biomolecular aggregate session M. Di Carlo (CNR-Palermo) and M. Digman (CA Univ. at Irvine) have interested the participants in the eVect on live cells of aggregation of beta-amyloid peptide and huntingtin, respectively. The session of Processes and mechanisms of the biological membranes has had its main points in the lectures concerning the studies of membrane permeability of ion channels (A. Moroni, Milano Univ.) or to antimicrobial peptides (G. Rispoli, Ferrara Univ.). For the session of Biomaterials and nano-biophysics O. Cavalleri (Genoa Univ.) and L. Cordone (Palermo Univ.) have discussed the possibility of a Wne control of protein folding/unfolding to build molecular nanostructures or the preservation properties of trehalose–water matrixes, respectively. The development of a framework for the registration of 3D imaging data for clinical heart investigations (R. Antolini, Trento Univ.) and the attempt to extend the results obtained on a charged, functionalized and patchy colloidal particles to globular protein solutions (F. Sciortino, Roma La Sapienza Univ.) have been discussed during the Theoretical and computational biophysics topic. In the session Photobiophysics and photoprocesses, T. Kottke (Bielefeld Univ.) has discussed the mechanism of blue light reception in plant and animal cryptochromes and D. Dell’Orco (Modena and Reggio Emilia Univ.) the molecular systems biology approaches to phototransduction in vertebrate rods. The investigation of a new biological scenario in which gene expression and regulation largely depend on a complex posttranscriptional network signals acting on mRNAs (A. Quattrone, Trento Univ.) and the relationship between structure and function in the P450 cytochrome involved in the erythromycin biosynthesis (B. Vallone, Roma La Sapienza Univ.) have been debated during the session regarding the Structure and function of proteins and nucleic acids. The biophysical mechanisms of regulation of CLC chloride channels and transporters (M. Pusch, Genoa CNR) and the application of optical and Xuorescence microscopies for dynamical tracking from cellular to molecular resolution (M. Faretta, Milan Eur. Inst. Oncol.) have been discussed during the Molecular and cellular biophysics session. The last day of the meeting, four young scientists have received the prize for their research: D. Giacomazza (&) Institute of Biophysics, National Research Council, Via U. La Malfa 153, Palermo, Italy e-mail: daniela.giacomazza@pa.ibf.cnr.it

Shedding Light on Animal Cryptochromes

Shedding Light on Animal Cryptochromes

Human and Drosophila Cryptochromes Are Light Activated by Flavin Photoreduction in Living Cells

Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non–light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.

Animal Type 1 Cryptochromes: ANALYSIS OF THE REDOX STATE OF THE FLAVIN COFACTOR BY SITE-DIRECTED MUTAGENESIS

It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, FAD(*-), in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FAD(ox) when purified, and the flavin is readily reduced to FAD(*-) by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of FAD(*-) in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that FAD(*-) represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs.

Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome

Cryptochromes are sensory blue light receptors mediating various responses in plants and animals. Studies on the mechanism of plant cryptochromes have been focused on the flowering plant Arabidopsis. In the genome of the unicellular green alga Chlamydomonas reinhardtii, a single plant cryptochrome, Chlamydomonas photolyase homologue 1 (CPH1), has been identified. The N-terminal 500 amino acids comprise the light-sensitive domain of CPH1 linked to a C-terminal extension of similar size. We have expressed the light-sensitive domain heterologously in Escherichia coli in high yield and purity. The 59-kDa protein bears exclusively flavin adenine dinucleotide in its oxidized state. Illumination with blue light induces formation of a neutral flavin radical with absorption maxima at 540 and 580 nm. The reaction proceeds aerobically even in the absence of an exogenous electron donor, which suggests that it reflects a physiological response. The process is completely reversible in the dark and exhibits a decay time constant of 200 s in the presence of oxygen. Binding of ATP strongly stabilizes the radical state after illumination and impedes the dark recovery. Thus, ATP binding has functional significance for plant cryptochromes and does not merely result from structural homology to DNA photolyase. The light-sensitive domain responds to illumination by an increase in phosphorylation. The autophosphorylation takes place although the protein is lacking its native C-terminal extension. This finding indicates that the extension is dispensable for autophosphorylation, despite the role it has been assigned in mediating signal transduction in Arabidopsis.

A Novel Photoreaction Mechanism for the Circadian Blue Light Photoreceptor Drosophila Cryptochrome

Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FAD(ox)) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FAD(ox) to the red anionic FAD(.) radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FAD(ox) corresponds to the ground state, whereas the FAD(.) radical represents the light-activated state that mediates resetting of the Drosophila circadian clock.

Action Spectrum of Drosophila Cryptochrome

Cryptochromes are a highly conserved class of UV-A/blue light photoreceptors. In Drosophila, cryptochrome is required for the normal entrainment of circadian rhythms to light dark cycles. The photocycle and molecular mechanism of animal cryptochrome photoreception are presently unknown. Drosophila cryptochrome undergoes light-dependent degradation when heterologously expressed in Schneider-2 cells. We have generated Drosophila luciferase-cryptochrome fusion proteins to more precisely monitor light-dependent cryptochrome degradation. We found that the luciferase-cryptochrome fusion protein undergoes light-dependent degradation with luciferase activity declining approximately 50% within 5 min of light exposure and approximately 85% within 1 h of light exposure. Degradation is inhibited by MG-132, consistent with a proteasomal degradation mechanism. Irradiance-response curves yield an action spectrum similar to absorption spectra for prokaryotic and eukaryotic cryptochromes with highest sensitivity in the UV-A. A luciferase-cryptochrome fusion protein lacking the terminal 15 amino acids is stably expressed in the dark but demonstrates increased sensitivity to light-induced degradation. The conferral of light-dependent degradation on a heterologous protein by fusion to cryptochrome may be a useful tool for probing protein function in cell expression systems.

Structure and Function of Animal Cryptochromes

Cryptochrome (CRY) is a photolyase-like flavoprotein with no DNA-repair activity but with known or presumed blue-light receptor function. Animal CRYs have DNA-binding and autokinase activities, and their flavin cofactor is reduced by photoinduced electron transfer. In Drosophila, CRY is a major circadian photoreceptor, and in mammals, the two CRY proteins are core components of the molecular clock and potential circadian photoreceptors. In mammals, CRYs participate in cell cycle regulation and the cellular response to DNA damage by controlling the expression of some cell cycle genes and by directly interacting with checkpoint proteins.

Light-induced Electron Transfer in Arabidopsis Cryptochrome-1 Correlates with in Vivo Function

Cryptochromes are blue light-activated photoreceptors found in multiple organisms with significant similarity to photolyases, a class of light-dependent DNA repair enzymes. Unlike photolyases, cryptochromes do not repair DNA and instead mediate blue light-dependent developmental, growth, and/or circadian responses by an as yet unknown mechanism of action. It has recently been shown that Arabidopsis cryptochrome-1 retains photolyase-like photoreduction of its flavin cofactor FAD by intraprotein electron transfer from tryptophan and tyrosine residues. Here we demonstrate that substitution of two conserved tryptophans that are constituents of the flavin-reducing electron transfer chain in Escherichia coli photolyase impairs light-induced electron transfer in the Arabidopsis cryptochrome-1 photoreceptor in vitro. Furthermore, we show that these substitutions result in marked reduction of light-activated autophosphorylation of cryptochrome-1 in vitro and of its photoreceptor function in vivo, consistent with biological relevance of the electron transfer reaction. These data support the possibility that light-induced flavin reduction via the tryptophan chain is the primary step in the signaling pathway of plant cryptochrome.

Role of Structural Plasticity in Signal Transduction by the Cryptochrome Blue-Light Photoreceptor

Cryptochromes are blue-light photoreceptors that regulate a variety of responses such as growth and circadian rhythms in organisms ranging from bacteria to humans. Cryptochromes share a high level of sequence identity with the light-activated DNA repair enzyme photolyase. Photolyase uses energy from blue light to repair UV-induced photoproducts in DNA through cyclic electron transfer between the catalytic flavin adenine dinucleotide cofactor and the damaged DNA. Cryptochromes lack DNA repair activity, and their mechanism of signal transduction is not known. It is hypothesized that a light-dependent signaling state in cryptochromes is created as a result of an intramolecular redox reaction, resulting in conformational rearrangement and effector binding. Plant and animal cryptochromes possess 30-250 amino acid carboxy-terminal extensions beyond the photolyase-homology region that have been shown to mediate phototransduction. We analyzed the structures of C-terminal domains from an animal and a plant cryptochrome by computational, biophysical, and biochemical methods and found these domains to be intrinsically unstructured. We show that the photolyase-homology region interacts with the C-terminal domain, inducing stable tertiary structure in the C-terminal domain. Importantly, we demonstrate a light-dependent conformational change in the C-terminal domain of Arabidopsis Cry1. Collectively, these findings provide the first biochemical evidence for the proposed conformational rearrangement of cryptochromes upon light exposure.

The cryptochromes

Cryptochromes are photoreceptors that regulate entrainment of the circadian clock by light in plants and animals. They are related to DNA photolyases and have similar three-dimensional structures, characterized by a α/β domain and a helical domain and including a chromophore, flavin adenine dinucleotide.

Novel ATP-binding and autophosphorylation activity associated with Arabidopsis and human cryptochrome-1.

Cryptochromes are blue-light photoreceptors sharing sequence similarity to photolyases, a class of flavoenzymes catalyzing repair of UV-damaged DNA via electron transfer mechanisms. Despite significant amino acid sequence similarity in both catalytic and cofactor-binding domains, cryptochromes lack DNA repair functions associated with photolyases, and the molecular mechanism involved in cryptochrome signaling remains obscure. Here, we report a novel ATP binding and autophosphorylation activity associated with Arabidopsis cry1 protein purified from a baculovirus expression system. Autophosphorylation occurs on serine residue(s) and is absent in preparations of cryptochrome depleted in flavin and/or misfolded. Autophosphorylation is stimulated by light in vitro and oxidizing agents that act as flavin antagonists prevent this stimulation. Human cry1 expressed in baculovirus likewise shows ATP binding and autophosphorylation activity, suggesting this novel enzymatic activity may be important to the mechanism of action of both plant and animal cryptochromes.

Molecular analysis of zebrafish photolyase/cryptochrome family: two types of cryptochromes present in zebrafish.

Cryptochromes (CRY), members of the DNA photolyase/cryptochrome protein family, regulate the circadian clock in animals and plants. Two types of animal CRYs are known, mammalian CRY and Drosophila CRY. Both CRYs participate in the regulation of circadian rhythm, but they have different light dependencies for their reactions and have different effects on the negative feedback loop which generates a circadian oscillation of gene expression. Mammalian CRYs act as a potent inhibitor of transcriptional activator whose reactions do not depend on light, but Drosophila CRY functions as a light-dependent suppressor of transcriptional inhibitor. We cloned seven zebrafish genes that carry members of the DNA photolyase/cryptochrome protein family; one (6-4)photolyase and six cry genes. A sequence analysis and determination of their in vitro functions showed that these zebrafish cry genes constitute two groups. One has a high sequence similarity to mammalian cry genes and inhibits CLOCK:BMAL1 mediated transcription. The other, which has a higher sequence similarity to the Drosophila cry gene rather than the mammalian cry genes, does not carry transcription inhibitor activity. The expressions of these cry genes oscillate in a circadian manner, but their patterns differ. These findings suggest that functionally diverse cry genes are present in zebrafish and each gene has different role in the molecular clock.

Blue-Light Photoreceptors in Higher Plants

In the past few years great progress has been made in identifying and characterizing plant photoreceptors active in the blue/UV-A regions of the spectrum. These photoreceptors include cryptochrome 1 and cryptochrome 2, which are similar in structure and chromophore composition to the prokaryotic DNA photolyases. However, they have a C-terminal extension that is not present in photolyases and lack photolyase activity. They are involved in regulation of cell elongation and in many other processes, including interfacing with circadian rhythms and activating gene transcription. Animal cryptochromes that play a photoreceptor role in circadian rhythms have also been characterized. Phototropin, the protein product of the NPH1 gene in Arabidopsis, likely serves as the photoreceptor for phototropism and appears to have no other role. A plasma membrane protein, it serves as photoreceptor, kinase, and substrate for light-activated phosphorylation. The carotenoid zeaxanthin may serve as the chromophore for a photoreceptor involved in blue-light-activated stomatal opening. The properties of these photoreceptors and some of the downstream events they are known to activate are discussed.