Opposing effects of an F-box protein and the HSP90 chaperone network on microtubule stability and neurite growth in Caenorhabditis elegans.

Molecular chaperones often work collaboratively with the ubiquitylation-proteasome system (UPS) to facilitate the degradation of misfolded proteins, which typically safeguards cellular differentiation and protects cells from stress. In this study, however, we report that the Hsp70/Hsp90 chaperone machinery and an F-box protein, MEC-15, have opposing effects on neuronal differentiation, and that the chaperones negatively regulate neuronal morphogenesis and functions. Using the touch receptor neurons (TRNs) of Caenorhabditis elegans, we find that mec-15(-) mutants display defects in microtubule formation, neurite growth, synaptic development and neuronal functions, and that these defects can be rescued by the loss of Hsp70/Hsp90 chaperones and co-chaperones. MEC-15 probably functions in a Skp-, Cullin- and F-box- containing complex to degrade DLK-1, which is an Hsp90 client protein stabilized by the chaperones. The abundance of DLK-1, and likely other Hsp90 substrates, is fine-tuned by the antagonism between MEC-15 and the chaperones; this antagonism regulates TRN development, as well as synaptic functions of GABAergic motor neurons. Therefore, a balance between the UPS and the chaperones tightly controls neuronal differentiation.

Inhibition of cell fate repressors secures the differentiation of the touch receptor neurons of Caenorhabditis elegans.

Terminal differentiation generates the specialized features and functions that allow postmitotic cells to acquire their distinguishing characteristics. This process is thought to be controlled by transcription factors called 'terminal selectors' that directly activate a set of downstream effector genes. In Caenorhabditis elegans, the differentiation of both the mechanosensory touch receptor neurons (TRNs) and the multidendritic nociceptor FLP neurons uses the terminal selectors UNC-86 and MEC-3. The FLP neurons fail to activate TRN genes, however, because a complex of two transcriptional repressors (EGL-44/EGL-46) prevents their expression. Here, we show that the ZEB family transcriptional factor ZAG-1 promotes TRN differentiation not by activating TRN genes but by preventing the expression of EGL-44/EGL-46. As EGL-44/EGL-46 also inhibits the production of ZAG-1, these proteins form a bistable, negative-feedback loop that regulates the choice between the two neuronal fates.

Neurosensory mechanotransduction.

Neurons that sense touch, sound and acceleration respond rapidly to specific mechanical signals. The proteins that transduce these signals and underlie these senses, however, are mostly unknown. Research over the past decade has suggested that members of three families of channel proteins are candidate transduction molecules. Current studies are directed towards characterizing these candidates, determining how they are mechanically gated and discovering new molecules that are involved in mechanical sensing.

A combinatorial regulatory signature controls terminal differentiation of the dopaminergic nervous system in C. elegans.

Terminal differentiation programs in the nervous system are encoded by cis-regulatory elements that control the expression of terminal features of individual neuron types. We decoded the regulatory information that controls the expression of five enzymes and transporters that define the terminal identity of all eight dopaminergic neurons in the nervous system of the Caenorhabditis elegans hermaphrodite. We show that the tightly coordinated, robust expression of these dopaminergic enzymes and transporters ("dopamine pathway") is ensured through a combinatorial cis-regulatory signature that is shared by all dopamine pathway genes. This signature is composed of an Ets domain-binding site, recognized by the previously described AST-1 Ets domain factor, and two distinct types of homeodomain-binding sites that act in a partially redundant manner. Through genetic screens, we identified the sole C. elegans Distalless/Dlx ortholog, ceh-43, as a factor that acts through one of the homeodomain sites to control both induction and maintenance of terminal dopaminergic fate. The second type of homeodomain site is a Pbx-type site, which is recognized in a partially redundant and neuron subtype-specific manner by two Pbx factors, ceh-20 and ceh-40, revealing novel roles of Pbx factors in the context of terminal neuron differentiation. Taken together, we revealed a specific regulatory signature and cognate, terminal selector-type transcription factors that define the entire dopaminergic nervous system of an animal. Dopaminergic neurons in the mouse olfactory bulb express a similar combinatorial transcription factor collective of Ets/Dlx/Pbx factors, suggesting deep phylogenetic conservation of dopaminergic regulatory programs.

A touching story.

A slide taped to a window at the Woods Hole Marine Biology Laboratory was my first introduction to the touch receptor neurons of the nematode Caenorhabditis elegans. Studying these cells as a postdoc with Sydney Brenner gave me a chance to work with John Sulston on a fascinating set of neurons. I would never have guessed then that 43 years later I would still be excited about learning their secrets.

GEFs and Rac GTPases control directional specificity of neurite extension along the anterior-posterior axis.

Although previous studies have identified many extracellular guidance molecules and intracellular signaling proteins that regulate axonal outgrowth and extension, most were conducted in the context of unidirectional neurite growth, in which the guidance cues either attract or repel growth cones. Very few studies addressed how intracellular signaling molecules differentially specify bidirectional outgrowth. Here, using the bipolar PLM neurons in Caenorhabditis elegans, we show that the guanine nucleotide exchange factors (GEFs) UNC-73/Trio and TIAM-1 promote anterior and posterior neurite extension, respectively. The Rac subfamily GTPases act downstream of the GEFs; CED-10/Rac1 is activated by TIAM-1, whereas CED-10 and MIG-2/RhoG act redundantly downstream of UNC-73. Moreover, these two pathways antagonize each other and thus regulate the directional bias of neuritogenesis. Our study suggests that directional specificity of neurite extension is conferred through the intracellular activation of distinct GEFs and Rac GTPases.

Regulation of the epithelial Na+ channel by paraoxonase-2.

Paraoxonase-2 (PON-2) is a membrane-bound lactonase with unique anti-oxidative and anti-atherosclerotic properties. PON-2 shares key structural elements with MEC-6, an endoplasmic reticulum-resident molecular chaperone in Caenorhabditis elegans MEC-6 modulates the expression of a mechanotransductive ion channel comprising MEC-4 and MEC-10 in touch-receptor neurons. Because pon-2 mRNA resides in multiple rat nephron segments, including the aldosterone-sensitive distal nephron where the epithelial Na+ channel (ENaC) is expressed, we hypothesized that PON-2 would similarly regulate ENaC expression. We observed PON-2 expression in aquaporin 2-positive principal cells of the distal nephron of adult human kidney. PON-2 also co-immunoprecipitated with ENaC when co-expressed in HEK293 cells. When PON-2 was co-expressed with ENaC in Xenopus oocytes, ENaC activity was reduced, reflecting a reduction in ENaC surface expression. MEC-6 also reduced ENaC activity when co-expressed in Xenopus oocytes. The PON-2 inhibitory effect was ENaC-specific, as PON-2 had no effect on functional expression of the renal outer medullary potassium channel. PON-2 did not alter the response of ENaC to extracellular Na+, mechanical shear stress, or α-chymotrypsin-mediated proteolysis, suggesting that PON-2 did not alter the regulation of ENaC by these factors. Together, our data suggest that PON-2 regulates ENaC activity by modulating its intracellular trafficking and surface expression.

Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans.

Wnt proteins regulate axonal outgrowth along the anterior-posterior axis, but the intracellular mechanisms that modulate the strength of Wnt signaling in axon guidance are largely unknown. Using the Caenorhabditis elegans mechanosensory PLM neurons, we found that posteriorly enriched LIN-44/Wnt acts as a repellent to promote anteriorly directed neurite outgrowth through the LIN-17/Frizzled receptor, instead of controlling neuronal polarity as previously thought. Dishevelled (Dsh) proteins DSH-1 and MIG-5 redundantly mediate the repulsive activity of the Wnt signals to induce anterior outgrowth, whereas DSH-1 also provides feedback inhibition to attenuate the signaling to allow posterior outgrowth against the Wnt gradient. This inhibitory function of DSH-1, which requires its dishevelled, Egl-10, and pleckstrin (DEP) domain, acts by promoting LIN-17 phosphorylation and is antagonized by planar cell polarity signaling components Van Gogh (VANG-1) and Prickle (PRKL-1). Our results suggest that Dsh proteins both respond to Wnt signals to shape neuronal projections and moderate its activity to fine-tune neuronal morphology.

Subunit composition of a DEG/ENaC mechanosensory channel of Caenorhabditis elegans.

Caenorhabditis elegans senses gentle touch in the six touch receptor neurons (TRNs) using a mechanotransduction complex that contains the pore-forming degenerin/epithelial sodium channel (DEG/ENaC) proteins MEC-4 and MEC-10. Past work has suggested these proteins interact with the paraoxonase-like MEC-6 and the cholesterol-binding stomatin-like MEC-2 proteins. Using single molecule optical imaging in Xenopus oocytes, we found that MEC-4 forms homotrimers and MEC-4 and MEC-10 form 4:4:10 heterotrimers. MEC-6 and MEC-2 do not associate tightly with these trimers and do not influence trimer stoichiometry, indicating that they are not part of the core channel transduction complex. Consistent with the in vitro data, MEC-10, but not MEC-6, formed puncta in TRN neurites that colocalize with MEC-4 when MEC-4 is overexpressed in the TRNs.

Regulation of mechanosensation in C. elegans through ubiquitination of the MEC-4 mechanotransduction channel.

In Caenorhabditis elegans, gentle touch is sensed by the anterior (ALM and AVM) and posterior (PLM) touch receptor neurons. Anterior, but not posterior, touch is affected by several stress conditions via the action of AKT kinases and the DAF-16/FOXO transcription factor. Here we show that a ubiquitination-dependent mechanism mediates such effects. AKT-1/AKT kinase and DAF-16 alter the transcription of mfb-1, which encodes an E3 ubiquitin ligase needed for the ubiquitination of the mechanosensory channel subunit MEC-4. Ubiquitination of MEC-4 reduces the amount of MEC-4 protein in the processes of ALM neurons and, consequently, the mechanoreceptor current. Even under nonstress conditions, differences in the amount of MFB-1 appear to cause the PLM neurons to be less sensitive to touch than the ALM neurons. These studies demonstrate that modulation of surface mechanoreceptors can regulate the sensitivity to mechanical signals.

Histone methylation restrains the expression of subtype-specific genes during terminal neuronal differentiation in Caenorhabditis elegans.

Although epigenetic control of stem cell fate choice is well established, little is known about epigenetic regulation of terminal neuronal differentiation. We found that some differences among the subtypes of Caenorhabditis elegans VC neurons, particularly the expression of the transcription factor gene unc-4, require histone modification, most likely H3K9 methylation. An EGF signal from the vulva alleviated the epigenetic repression of unc-4 in vulval VC neurons but not the more distant nonvulval VC cells, which kept unc-4 silenced. Loss of the H3K9 methyltransferase MET-2 or H3K9me2/3 binding proteins HPL-2 and LIN-61 or a novel chromodomain protein CEC-3 caused ectopic unc-4 expression in all VC neurons. Downstream of the EGF signaling in vulval VC neurons, the transcription factor LIN-11 and histone demethylases removed the suppressive histone marks and derepressed unc-4. Behaviorally, expression of UNC-4 in all the VC neurons caused an imbalance in the egg-laying circuit. Thus, epigenetic mechanisms help establish subtype-specific gene expression, which are needed for optimal activity of a neural circuit.

Modulation of C. elegans touch sensitivity is integrated at multiple levels.

Sensory systems can adapt to different environmental signals. Here we identify four conditions that modulate anterior touch sensitivity in Caenorhabditis elegans after several hours and demonstrate that such sensory modulation is integrated at multiple levels to produce a single output. Prolonged vibration involving integrin signaling directly sensitizes the touch receptor neurons (TRNs). In contrast, hypoxia, the dauer state, and high salt reduce touch sensitivity by preventing the release of long-range neuroregulators, including two insulin-like proteins. Integration of these latter inputs occurs at upstream neurohormonal cells and at the insulin signaling cascade within the TRNs. These signals and those from integrin signaling converge to modulate touch sensitivity by regulating AKT kinases and DAF-16/FOXO. Thus, activation of either the integrin or insulin pathways can compensate for defects in the other pathway. This modulatory system integrates conflicting signals from different modalities, and adapts touch sensitivity to both mechanical and non-mechanical conditions.

A disease-causing mutation illuminates the protein membrane topology of the kidney-expressed prohibitin homology (PHB) domain protein podocin.

Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocin(P118L)) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2(P134S)). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin(P118L) and MEC-2(P134S) did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.

Shared gene expression in distinct neurons expressing common selector genes.

Expression of the mec-3/unc-86 selector gene complex induces the differentiation of the touch receptor neurons (TRNs) of Caenorhabditis elegans. These genes are also expressed in another set of embryonically derived mechanosensory neurons, the FLP neurons, but these cells do not share obvious TRN traits or proteins. We have identified ~300 genes in each cell type that are up-regulated at least threefold using DNA microarrays. Twenty-three percent of these genes are up-regulated in both cells. Surprisingly, some of the common genes had previously been identified as TRN-specific. Although the FLP neurons contain low amounts of the mRNAs for these TRN genes, they do not have detectable proteins. These results suggest that transcription control is relatively inexact but that these apparent errors of transcription are tolerated and do not alter cell fate. Previous studies showed that loss of the EGL-44 and EGL-46 transcription factors cause the FLP neurons to acquire TRN-like traits. Here, we show that similar changes occur (e.g., the expression of both the TRN mRNAs and proteins) when the FLP neurons ectopically express the auxiliary transcription factor ALR-1 (Aristaless related), which ensures, but does not direct, TRN differentiation. Thus, the FLP neurons can acquire a TRN-like fate but use multiple levels of regulation to ensure they do not. Our data indicate that expression of common master regulators in different cell types can result in inappropriate expression of effector genes. This misexpression makes these cells vulnerable to influences that could cause them to acquire alternative fates.

Caenorhabditis elegans aristaless/Arx gene alr-1 restricts variable gene expression.

Variable expressivity of mutant phenotypes in genetically identical individuals is a phenomenon widely reported but poorly understood. For example, mutations in the gene encoding the transcription factor ALR-1 in Caenorhabditis elegans result in variable touch receptor neuron (TRN) function. Using single-molecule in situ hybridization, we demonstrate that this phenotypic variability reflects enhanced variability in the expression of the selector gene mec-3, which is needed, together with unc-86, for the differentiation of the TRNs. In a yeast expression system, ALR-1 enhances MEC-3/UNC-86-dependent transcription from the mec-3 promoter, showing that ALR-1 can enhance bulk mec-3 expression. We show that, due to stochastic fluctuations, autoregulation of mec-3 is not sufficient for TRN differentiation; ALR-1 provides a second positive feedback loop that increases mec-3 expression, by restricting variability, and thus ensures TRN differentiation. Our results link fluctuations in gene expression to phenotypic variability, which is seen in many mutant strains, and provide an explicit demonstration of how variable gene expression can be curtailed in developing cells to ensure their differentiation. Because ALR-1 and similar proteins (Drosophila Aristaless and human ARX) are needed for the expression of other transcription factors, we propose that proteins in this family may act to ensure differentiation more generally.

Microtubule depolymerization in Caenorhabditis elegans touch receptor neurons reduces gene expression through a p38 MAPK pathway.

Microtubules are integral to neuronal development and function. They endow cells with polarity, shape, and structure, and their extensive surface area provides substrates for intracellular trafficking and scaffolds for signaling molecules. Consequently, microtubule polymerization dynamics affect not only structural features of the cell but also the subcellular localization of proteins that can trigger intracellular signaling events. In the nematode Caenorhabditis elegans, the processes of touch receptor neurons are filled with a bundle of specialized large-diameter microtubules. We find that conditions that disrupt these microtubules (loss of either the MEC-7 ß-tubulin or MEC-12 α-tubulin or growth in 1 mM colchicine) cause a general reduction in touch receptor neuron (TRN) protein levels. This reduction requires a p38 MAPK pathway (DLK-1, MKK-4, and PMK-3) and the transcription factor CEBP-1. Cells may use this feedback pathway that couples microtubule state and MAPK activation to regulate cellular functions.