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Organisms rely on chemical cues in their environment to indicate the presence or absence of food, reproductive partners, predators, or other harmful stimuli. In the nematode Caenorhabditis elegans, the bilaterally symmetric pair of ASH sensory neurons serves as the primary nociceptors. ASH activation by aversive stimuli leads to backward locomotion and stimulus avoidance. We previously reported a role for guanylyl cyclases in dampening nociceptive sensitivity that requires an innexin-based gap junction network to pass cGMP between neurons. Here, we report that animals lacking function of the gap junction component INX-20 are hypersensitive in their behavioral response to both soluble and volatile chemical stimuli that signal through G protein-coupled receptor pathways in ASH. We find that expressing inx-20in the ADL and AFD sensory neurons is sufficient to dampen ASH sensitivity, which is supported by new expression analysis of endogenous INX-20 tagged with mCherry via the CRISPR-Cas9 system. Although ADL does not form gap junctions directly with ASH, it does so via gap junctions with the interneuron RMG and the sensory neuron ASK. Ablating either ADL or RMG and ASK also resulted in nociceptive hypersensitivity, suggesting an important role for RMG/ASK downstream of ADL in the ASH modulatory circuit. This work adds to our growing understanding of the repertoire of ways by which ASH activity is regulated via its connectivity to other neurons and identifies a previously unknown role for ADL and RMG in the modulation of aversive behavior.

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 vitroand 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-3β (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 Xenopusembryos and suggest that GSK-3 regulates eye development through a non-Wnt pathway.

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.

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 elegansan 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. elegansto chemicals, including pheromones. The majority of identified 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. eleganswill 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.

Methylation of arginine residues in dopamine receptors in humans and worms promotes signaling and functional responses.