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Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein's function. We present Caenorhabditis elegans light-induced coclustering (CeLINC), an optical binary protein-protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Colocalization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome. [ABSTRACT FROM AUTHOR]

Arabidopsis cryptochrome 2 (CRY2) and FLAVIN-BINDING, KELCH REPEAT, and 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 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 noncanonical 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 the transcription of the GI gene. FKF1 is required for CIB3 to promote flowering and enhances the CIB1–CIB3 interaction in response to blue light. [ABSTRACT FROM AUTHOR]

Disruption of circadian rhythms is associated with aberrant trophoblast migration and invasion in recurrent spontaneous abortion (RSA). This study aims to explore the functional role and the mechanisms of cryptochrome 2 (CRY2), a fundamental component of the circadian clock, in regulating trophoblast migration and invasion. Human extravillous trophoblast cell line HTR-8/SVneo was used as a cell model. Cell migration and invasion were examined using wound healing assay and Transwell assay, respectively. The mRNA and protein levels were determined using quantitative real-time polymerase chain reaction and western blot, respectively. Luciferase reporter assay and chromatin immunoprecipitation assay were performed to explore the interaction between c-Myc to the brain and muscle ARNT-like protein 1 (BMAL1) promoter. CRY2 was highly expressed in human villous specimens of RSA. Furthermore, CRY2 overexpression impaired migration and invasion in HTR-8/SVneo cells, whereas CRY2 knockdown yielded the opposite results. Mechanistically, c-Myc bound to the BMAL1 promoter and induced BMAL1 transcription, both of which further activated matrix metalloproteinase 2/9 (MMP2/9) and facilitated migration and invasion in HTR-8/SVneo cells. CRY2 inhibited c-Myc-BMAL1 pathway and impaired migration and invasion of HTR-8/SVneo cells. Collectively, these findings demonstrate that CRY2 suppresses trophoblast migration and invasion via inhibiting c-Myc-BMAL1-MMP2/9 pathway. [ABSTRACT FROM AUTHOR]

The soil-borne fungal pathogen Verticillium dahliae infects a wide range of dicotyledonous plants including cotton, tobacco, and Arabidopsis. Among the effector proteins secreted by V. dahliae, the 16 kDa PevD1 induces a hypersensitive response in tobacco. Here we report the high-resolution structure of PevD1 with folds resembling a C2 domain-like structure with a calcium ion bound to the C-terminal acidic pocket. A yeast two-hybrid screen, designed to probe for molecular functions of PevD1, identified Arabidopsis asparagine-rich protein (NRP) as the interacting partner of PevD1. Extending the pathway of V. dahliae effects, which include induction of early flowering in cotton and Arabidopsis, NRP was found to interact with cryptochrome 2 (CRY2), leading to increased cytoplasmic accumulation of CRY2 in a blue light-independent manner. Further physiological and genetic evidence suggests that PevD1 indirectly activates CRY2 by antagonizing NRP functions. The promotion of CRY2-mediated flowering by a fungal effector outlines a novel pathway by which an external stimulus is recognized and transferred in changing a developmental program. [ABSTRACT FROM AUTHOR]

Cryptochromes are blue light receptors regulated by lightdependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasomedependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the nonpermissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases. [ABSTRACT FROM AUTHOR]

It is recognized that the behavioral rhythms of organisms are controlled by the circadian clock, while the reverse direction, i.e. whether changes in physiology and behavior react to the internal rhythms, is unclear. Cryptochromes (CRYs) are photolyase-like flavoproteins with blue-light receptor function and other functions on circadian clock and migration in animals. Here, we cloned the full-length cDNA of CRY1 and CRY2 in Spodoptera litura (Fabricius, 1775) (Lepidoptera: Noctuidae). Sl -CRYs show high similarity to orthologs from other insects, and their conserved regions contain a DNA photolyase domain and a FAD-binding seven domain. The expression levels of both genes were relatively low during the larval stage, which increased during the pupal stage and then peaked at the adult stage. The expression of Sl-CRY1 and Sl-CRY2 showed differences between males and females and between scotophase and photophase. Further, our study demonstrated that copulation has a significant effect on the expression of Sl-CRYs. More interestingly, the changes in the expression of Sl-CRY1 and Sl-CRY2 due to copulation showed the same trend in both sexes, in which the expression levels of both genes in copulated males and females decreased in the subsequent scotophase after copulation and then increased significantly in the following photophase. Considering the nature of the dramatic changes in reproductive behavior and physiology after copulation in S. litura, we propose that the changes in the expression of Sl-CRYs after copulation could have some function in the reproductive process. [ABSTRACT FROM AUTHOR]