C-Terminal Extension of a Plant Cryptochrome Dissociates from the ß-Sheet of the Flavin-Binding Domain.

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from Chlamydomonas reinhardtii in living bacterial cells, because the receptor degraded upon isolation. We demonstrate a stabilization of the flavin neutral radical as photoproduct and of the resulting ß-sheet reorganization by binding of cellular ATP. Comparison between light-induced structural responses of full-length pCRY and PHR reveals a downshift in frequency of the ß-sheet signal, implying an association of the CCT close to the only ß-sheet of the PHR in the dark. We provide a missing link in activation of plant cryptochromes after flavin photoreduction by indicating that ß-sheet reorganization causes the CCT release and restructuring.

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

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.

Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools.

In this work we evaluate the interaction of two optogenetic protein variants (CIB1, CIBN) with their complementary protein CRY2 by single-molecule tools in cell-free extracts. After validating the blue light induced co-localization of CRY2 and CIB1/N by Förster resonance energy transfer (FRET) in live cells, a fluorescence correlation spectroscopy (FCS) based method was developed to quantitatively determine the in vitro association of the extracted proteins. Our experiments suggest that CIB1, in comparison with CIBN, possesses a better coupling efficiency with CRY2 due to its intact protein structure and lower diffusion rate within 300s detection window.

Cryptochrome in sponges: a key molecule linking photoreception with phototransduction.

Sponges (phylum: Porifera) react to external light or mechanical signals with contractile or metabolic reactions and are devoid of any nervous or muscular system. Furthermore, elements of a photoreception/phototransduction system exist in those animals. Recently, a cryptochrome-based photoreceptor system has been discovered in the demosponge. The assumption that in sponges the siliceous skeleton acts as a substitution for the lack of a nervous system and allows light signals to be transmitted through its glass fiber network is supported by the findings that the first spicules are efficient light waveguides and the second sponges have the enzymatic machinery for the generation of light. Now, we have identified/cloned in Suberites domuncula two additional potential molecules of the sponge cryptochrome photoreception system, the guanine nucleotide-binding protein ß subunit, related to ß-transducin, and the nitric oxide synthase (NOS)-interacting protein. Cryptochrome and NOSIP are light-inducible genes. The studies show that the NOS inhibitor L-NMMA impairs both morphogenesis and motility of the cells. Finally, we report that the function of primmorphs to produce reactive nitrogen species can be abolished by a NOS inhibitor. We propose that the sponge cryptochrome-based photoreception system, through which photon signals are converted into radicals, is coupled to the NOS apparatus.

Regulation of core clock genes in human islets.

Nearly all mammalian cells express a set of genes known as clock genes. These regulate the circadian rhythm of cellular processes by means of negative and positive autoregulatory feedback loops of transcription and translation. Recent genomewide association studies have demonstrated an association between a polymorphism near the circadian clock gene CRY2 and elevated fasting glucose. To determine whether clock genes could play a pathogenetic role in the disease, we examined messenger RNA (mRNA) expression of core clock genes in human islets from donors with or without type 2 diabetes mellitus. Microarray and quantitative real-time polymerase chain reaction analyses were used to assess expression of the core clock genes CLOCK, BMAL-1, PER1 to 3, and CRY1 and 2 in human islets. Insulin secretion and insulin content in human islets were measured by radioimmunoassay. The mRNA levels of PER2, PER3, and CRY2 were significantly lower in islets from donors with type 2 diabetes mellitus. To investigate the functional relevance of these clock genes, we correlated their expression to insulin content and glycated hemoglobin levels: mRNA levels of PER2 (ρ = 0.33, P = .012), PER3 (ρ = 0.30, P = .023), and CRY2 (ρ = 0.37, P = .0047) correlated positively with insulin content. Of these genes, expression of PER3 and CRY2 correlated negatively with glycated hemoglobin levels (ρ = -0.44, P = .0012; ρ = -0.28, P = .042). Furthermore, in an in vitro model mimicking pathogenetic conditions, the PER3 mRNA level was reduced in human islets exposed to 16.7 mmol/L glucose per 1 mmol/L palmitate for 48 hours (P = .003). Core clock genes are regulated in human islets. The data suggest that perturbations of circadian clock components may contribute to islet pathophysiology in human type 2 diabetes mellitus.