Predictive Spatiotemporal Manipulation of Signaling Perturbations Using Optogenetics

Recently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 μm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability.     

Auxiliary Ca2+ binding sites can influence the structure of CIB1

Recent X-ray crystal structures and solution NMR spectroscopy data for calcium- and integrin-binding protein 1 (CIB1) have all revealed a common EF-hand domain structure for the protein. However, the orientation of the two protein domains, the oligomerization state, and the conformations of the N- and C-terminal extensions differ among the structures. In this study, we examine whether the binding of glutathione or auxiliary Ca²⁺ ions as observed in the crystal structures, occur in solution, and whether these interactions can influence the structure or dimerization of CIB1. In addition, we test the potential phosphatase activity of CIB1, which was hypothesized based on the glutathione binding site geometry observed in one of the crystal structures of the protein. Biophysical and biochemical experiments failed to detect glutathione binding, protein dimerization, or phosphatase activity for CIB1 under several solution conditions. However, our data identify low affinity (K_d, 10⁻²M) Ca²⁺ binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high (300 mM) Ca²⁺ crystallization conditions. In addition to providing a rationale for differences amongst the various solution and crystal structures of CIB1, our results show that the impact of low affinity Ca²⁺ binding events should be considered when analyzing and interpreting protein crystallographic structures determined in the presence of very high Ca²⁺ concentrations.     

Molecular Genetics of Cryptopleurine Resistance in Saccharomyces Cerevisiae: Expression of a Ribosomal Protein Gene Family

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-δ1::URA3 null allele is viable, cryptopleurine sensitive (Cry(S)), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage λ, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-δ2::TRP1 cry2-δ1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into Cry(R) yeast of genotype cry1 CRY2 confers a Cry(S) phenotype. Transformation of these Cry(R) yeast with CRY2 on a low copy CEN plasmid does not confer a Cry(S) phenotype. (2) Haploids containing the cry1-δ2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-δ1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of β-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the Cry(R) phenotype of cry2 mutants is only expressed in strains containing a cry1-δ null allele.     

CUL4-DDB1-CDT2 E3 Ligase Regulates the Molecular Clock Activity by Promoting Ubiquitination-Dependent Degradation of the Mammalian CRY1

The CUL4-DDB1 E3 ligase complex serves as a critical regulator in various cellular processes, including cell proliferation, DNA damage repair, and cell cycle progression. However, whether this E3 ligase complex regulates clock protein turnover and the molecular clock activity in mammalian cells is unknown. Here we show that CUL4-DDB1-CDT2 E3 ligase ubiquitinates CRY1 and promotes its degradation both in vitro and in vivo. Depletion of the major components of this E3 ligase complex, including Ddb1, Cdt2, and Cdt2-cofactor Pcna, leads to CRY1 stabilization in cultured cells or in the mouse liver. CUL4A-DDB1-CDT2 E3 ligase targets lysine 585 within the C-terminal region of CRY1 protein, shown by the CRY1 585KA mutant’s resistance to ubiquitination and degradation mediated by the CUL4A-DDB1 complex. Surprisingly, both depletion of Ddb1 and over-expression of Cry1-585KA mutant enhance the oscillatory amplitude of the Bmal1 promoter activity without altering its period length, suggesting that CUL4A-DDB1-CDT2 E3 targets CRY1 for degradation and reduces the circadian amplitude. All together, we uncovered a novel biological role for CUL4A-DDB1-CDT2 E3 ligase that regulates molecular circadian behaviors via promoting ubiquitination-dependent degradation of CRY1.     

Migration of the Fungal Protein Cryptogein within Tobacco Plants

Cryptogein (CRY), a protein secreted by Phytophthora cryptogea, causes necrosis on tobacco (Nicotiana tabacum) plants at the site of application (the stem or the roots) and also on distant leaves. Autoradiography of plantlets after root absorption of radioiodinated CRY demonstrated a rapid migration of the label to the leaf lamina via the veins. Using an anti-CRY antiserum, a CRY-related antigen was detected in the stem and leaves of CRY-treated plants at a distance from the site of application. This antigen had the same molecular weight as CRY and was detected in the leaves as early as 1 hour after stem treatment, i.e. long before necrosis was detectable. The antigen was also detected in plants inoculated with P. cryptogea. The distant location of the necrosis induced by the fungus or by CRY can be ascribed to the migration of this protein, which is toxic to tobacco cells. It is proposed that CRY, which also elicits defense reactions in tobacco, might contribute to the hypersensitive response of tobacco to P. cryptogea.     

A Novel C-Terminal CIB2 (Calcium and Integrin Binding Protein 2) Mutation Associated with Non-Syndromic Hearing Loss in a Hispanic Family

Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556C>T; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca²⁺ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing.     

Formation of Nuclear Bodies of Arabidopsis CRY2 in Response to Blue Light Is Associated with Its Blue Light–Dependent Degradation

Arabidopsis thaliana cryptochrome 2 (CRY2) mediates photoperiodic promotion of floral initiation and blue light inhibition of hypocotyl elongation. It has been hypothesized that photoexcitation derepresses CRY2 by disengaging its C-terminal domain from the N-terminal PHR domain. To test this hypothesis, we analyzed activities of CRY2 fused to green fluorescent protein (GFP) at either the N terminus (GFP-CRY2) or the C terminus (CRY2-GFP). While GFP-CRY2 exerts light-dependent biochemical and physiological activities similar to those of the endogenous CRY2, CRY2-GFP showed constitutive biochemical and physiological activities. CRY2-GFP is constitutively phosphorylated, it promotes deetiolation in both dark and light, and it activates floral initiation in both long-day and short-day photoperiods. These results are consistent with the hypothesis that photoexcited CRY2 disengages its C-terminal domain from the PHR domain to become active. Surprisingly, we found that CRY2-GFP, but not GFP-CRY2, formed distinct nuclear bodies in response to blue light. Compared with GFP-CRY2 or the endogenous CRY2, CRY2-GFP degradation was significantly retarded in response to blue light, suggesting that the nuclear bodies may result from accumulation of photoexcited CRY2-GFP waiting to be degraded. Consistent with this interpretation, we showed that both GFP-CRY2 and endogenous CRY2 formed nuclear bodies in the presence of the 26S-proteasome inhibitors that block blue light–dependent CRY2 degradation.     

Arabidopsis Casein Kinase1 Proteins CK1.3 and CK1.4 Phosphorylate Cryptochrome2 to Regulate Blue Light Signaling

Casein kinase1 (CK1) plays crucial roles in regulating growth and development via phosphorylating various substrates throughout the eukaryote kingdom. Blue light is crucial for normal growth of both plants and animals, and blue light receptor cryptochrome2 (CRY2) undergoes blue light–dependent phosphorylation and degradation in planta. To study the function of plant CK1s, systematic genetic analysis showed that deficiency of two paralogous Arabidopsis thaliana CK1s, CK1.3 and CK1.4, caused shortened hypocotyls, especially under blue light, while overexpression of either CK1.3 or CK1.4 resulted in the insensitive response to blue light and delayed flowering under long-day conditions. CK1.3 or CK1.4 act dependently on CRY2, and overexpression of CK1.3 or CK1.4 significantly suppresses the hypersensitive response to blue light by CRY2 overexpression. Biochemical studies showed that CK1.3 and CK1.4 directly phosphorylate CRY2 at Ser-587 and Thr-603 in vitro and negatively regulate CRY2 stability in planta, which are stimulated by blue light, further confirming the crucial roles of CK1.3 and CK1.4 in blue light responses through phosphorylating CRY2. Interestingly, expression of CK1.3 and CK1.4 is stimulated by blue light and feedback regulated by CRY2-mediated signaling. These results provide direct evidence for CRY2 phosphorylation and informative clues on the mechanisms of CRY2-mediated light responses.     

CRY2 Genetic Variants Associate with Dysthymia

People with mood disorders often have disruptions in their circadian rhythms. Recent molecular genetics has linked circadian clock genes to mood disorders. Our objective was to study two core circadian clock genes, CRY1 and CRY2 as well as TTC1 that interacts with CRY2, in relation to depressive and anxiety disorders. Of these three genes, 48 single-nucleotide polymorphisms (SNPs) whose selection was based on the linkage disequilibrium and potential functionality were genotyped in 5910 individuals from a nationwide population-based sample. The diagnoses of major depressive disorder, dysthymia and anxiety disorders were assessed with a structured interview (M-CIDI). In addition, the participants filled in self-report questionnaires on depressive and anxiety symptoms. Logistic and linear regression models were used to analyze the associations of the SNPs with the phenotypes. Four CRY2 genetic variants (rs10838524, rs7121611, rs7945565, rs1401419) associated significantly with dysthymia (false discovery rate q<0.05). This finding together with earlier CRY2 associations with winter depression and with bipolar type 1 disorder supports the view that CRY2 gene has a role in mood disorders.     

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins regulate gibberellin signaling and photomorphogenesis in Arabidopsis

Cryptochromes are blue light photoreceptors that mediate various light responses in plants and mammals. In Arabidopsis (Arabidopsis thaliana), cryptochrome 1 (CRY1) mediates blue light-induced photomorphogenesis, which is characterized by reduced hypocotyl elongation and enhanced anthocyanin production, whereas gibberellin (GA) signaling mediated by the GA receptor GA-INSENSITIVE DWARF1 (GID1) and DELLA proteins promotes hypocotyl elongation and inhibits anthocyanin accumulation. Whether CRY1 control of photomorphogenesis involves regulation of GA signaling is largely unknown. Here, we show that CRY1 signaling involves the inhibition of GA signaling through repression of GA-induced degradation of DELLA proteins. CRY1 physically interacts with DELLA proteins in a blue light-dependent manner, leading to their dissociation from SLEEPY1 (SLY1) and the inhibition of their ubiquitination. Moreover, CRY1 interacts directly with GID1 in a blue light-dependent but GA-independent manner, leading to the inhibition of the interaction between GID1 with DELLA proteins. These findings suggest that CRY1 controls photomorphogenesis through inhibition of GA-induced degradation of DELLA proteins and GA signaling, which is mediated by CRY1 inhibition of the interactions of DELLA proteins with GID1 and SCF^SLY1, respectively.

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins inhibit gibberellin (GA)-induced degradation of DELLA proteins to regulate GA signaling and photomorphogenesis.     

Nuclear localization of the Arabidopsis blue light receptor cryptochrome 2

The cryptochrome blue light photoreceptor family of Arabidopsis thaliana consists of two members, CRY1 and CRY2 (PHH1). CRY2 contains a putative nuclear localization signal (NLS) within its C-terminal region. We examined whether CRY2 is localized in the nucleus and whether the C-terminal region of CRY2 is involved in nuclear targeting. Total cellular and nuclear protein extracts from Arabidopsis were subjected to immunoblot analysis with CRY2-specific antibodies. Strong CRY2 signals were obtained in the nuclear fraction. Fusion proteins consisting of the green fluorescent protein (GFP) and different fragments of CRY2 were expressed in parsley protoplasts and the localization of the fusion proteins was determined by fluorescence and confocal laser scanning microscopy. GFP-fusions containing the entire CRY2 protein or its C-terminal region were found exclusively in the nucleus. We conclude from these results that CRY2 is localized in the nucleus and that nuclear localization is mediated by the C-terminal region of CRY2.