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cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli.

GBP is an important maternal protein that promotes the development of a dorso-ventral axis in developing Xenopus embryo. GBP stabilizes beta-catenin by binding kinase Xgsk-3. This interaction inhibits the phosphorylation of beta-catenin. The accumulation of stable beta-catenin in the dorsal region of the embryo is responsible for the development of the dorsal axis. Injection of GBP at different regions of the embryo induces the development of ectopic axis.

Chemosensory neurons translate perception of external chemical cues, including odorants, tastants, and pheromones, into information that drives attraction or avoidance motor programs. In the laboratory, robust behavioral assays, coupled with powerful genetic, molecular and optical tools, have made Caenorhabditis elegans an ideal experimental system in which to dissect the contributions of individual genes and neurons to ethologically relevant chemosensory behaviors. Here, we review current knowledge of the neurons, signal transduction molecules and regulatory mechanisms that underlie the response of C. elegans to chemicals, including pheromones. The majority of identi- fied molecules and pathways share remarkable homology with sensory mechanisms in other organisms. With the development of new tools and technologies, we anticipate that continued study of chemosensory signal transduction and processing in C. elegans will yield additional new insights into the mechanisms by which this animal is able to detect and discriminate among thousands of chemical cues with a limited sensory neuron repertoire. [ABSTRACT FROM AUTHOR]

The Wnt signal transduction pathway is important in a variety of developmental processes, including establishment of the early dorsal signaling center in Xenopus. A large body of evidence indicates that this dorsal organizing center functions to locally inhibit GSK-3 and thus upregulate [Beta]-catenin. Along with transcription factors of the Lef/Tcf family, [Beta]-catenin then activates the transcription of dorsal-specific genes. It now appears that multiple proteins with opposing effects converge on GSK-3 to regulate its activity and thus cytoplasmic [Beta]-catenin levels. Specifically, GSK-3 is normally part of a large protein complex that includes Axin, and this complex promotes GSK-3-mediated phosphorylation and degradation of [Beta]-catenin. However, following fertilization GSK-3 is inhibited dorsally, in part by GBP (GSK-3-binding protein). Since GBP and Axin compete for binding to GSK-3, we have screened for mutations in GSK-3 that eliminate binding to either GBP or Axin, but not both. We are using these mutants to further analyze the role of GSK-3, GBP, and Axin in early Xenopus development.

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity. [ABSTRACT FROM AUTHOR]

Recently, optical stimulation has begun to unravel the neuronal processing that controls certain animal behaviours. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Here, we show an approach based on radio-frequency magnetic-field heating of nanoparticles to remotely activate temperature-sensitive cation channels in cells. Superparamagnetic ferrite nanoparticles were targeted to specific proteins on the plasma membrane of cells expressing TRPV1, and heated by a radio-frequency magnetic field. Using fluorophores as molecular thermometers, we show that the induced temperature increase is highly localized. Thermal activation of the channels triggers action potentials in cultured neurons without observable toxic effects. This approach can be adapted to stimulate other cell types and, moreover, may be used to remotely manipulate other cellular machinery for novel therapeutics. [ABSTRACT FROM AUTHOR]

Accumulation of the Wnt pathway effector β-catenin is a hallmark of a number of cancers, including colon cancer. As β-catenin accumulates in the cell, it forms a complex with Tcf family transcription factors and activates the transcription of several critical genes involved in cell proliferation. Because Tcf4 is the predominant Tcf factor present in colon cancer cells, drugs that specifically disrupt the β-catenin?Tcf4 complex could be useful in treating colon cancers. Earlier structural and biochemical studies demonstrated that the central region of the β-catenin binding domain of Tcf is essential for anchoring Tcf to β-catenin via two conserved lysines in β-catenin (called the charged 'buttons'). Here we report the crystal structure of a β-catenin?Tcf4 complex at 2.0 Å resolution. Our structural and mutagenesis studies show that Tcf4 docks specifically to β-catenin using several distinct conformations in its essential central region. These conformations allow different glutamate residues in the central region of Tcf4 to form a salt bridge with the same critical charged button, Lys 312 of β-catenin. We propose that this interaction may be the first event in β-catenin?Tcf4 recognition. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors are 7-pass transmembrane receptors that couple to heterotrimeric G proteins to mediate cellular responses to a diverse array of stimuli. Understanding the mechanisms that regulate G protein-coupled receptors is crucial to manipulating their signaling for therapeutic benefit. One key regulatory mechanism that contributes to the functional diversity of many signaling proteins is post-translational modification. Whereas phosphorylation remains the best studied of such modifications, arginine methylation by protein arginine methyltransferases is emerging as a key regulator of protein function. We previously published the first functional evidence that arginine methylation of G protein-coupled receptors modulates their signaling. We report here a third receptor that is regulated by arginine methylation, the Caenorhabditis elegans SER-2 tyramine receptor. We show that arginines within a putative methylation motif in the third intracellular loop of SER-2 are methylated by PRMT5 in vitro. Our data also suggest that this modification enhances SER-2 signaling in vivo to modulate animal behavior. The identification of a third G protein-coupled receptor to be functionally regulated by arginine methylation suggests that this post-translational modification may be utilized to regulate signaling through a broad array of G protein-coupled receptors. [ABSTRACT FROM AUTHOR]

Although the affinity optimization of protein binders is straightforward, engineering epitope specificity is more challenging. Targeting a specific surface patch is important because the biological relevance of protein binders depends on how they interact with the target. They are particularly useful to test hypotheses motivated by biochemical and structural studies. We used yeast display to engineer monobodies that bind a defined surface patch on the mitogen activated protein kinase (MAPK) Erk-2. The targeted area ("CD" domain) is known to control the specificity and catalytic efficiency of phosphorylation by the kinase by binding a linear peptide ("D" peptide) on substrates and regulators. An inhibitor of the interaction should thus be useful for regulating Erk-2 signaling in vivo. Although the CD domain constitutes only a small percentage of the surface area of the enzyme (~5%), sorting a yeast displayed monobody library with wild type (wt) Erk-2 and a rationally designed mutant led to isolation of high affinity clones with desired epitope specificity. The engineered binders inhibited the activity of Erk-2 in vitro and in mammalian cells. Furthermore, they specifically inhibited the activity of Erk-2 orthologs in yeast and suppressed a mutant phenotype in round worms caused by overactive MAPK signaling. The study therefore shows that positive and negative screening can be used to bias the evolution of epitope specificity and predictably design inhibitors of biologically relevant protein-protein interaction.

Glycogen synthase kinase-3 beta (GSK-3) is a key downstream target of Wnt signaling and is regulated by its interactions with activating and inhibitory proteins. We and others have shown that GSK-3 activity toward non-primed substrates is regulated in part through a competition between its activating (Axin) and inhibitory (GBP/FRAT) binding partners. Here we use a reverse two-hybrid screen to identify mutations in GSK-3 that alter binding to GBP and Axin. We find that these mutations overlap and propose that GBP and Axin compete for binding to the same region of GSK-3. We use these mutations to examine the ability of GSK-3 to block eye development in Xenopus embryos and suggest that GSK-3 regulates eye development through a non-Wnt pathway.