Epithelial UNC-23 limits mechanical stress to maintain glia-neuron architecture in C. elegans.

For an organ to maintain correct architecture and function, its diverse cellular components must coordinate their size and shape. Although cell-intrinsic mechanisms driving homotypic cell-cell coordination are known, it is unclear how cell shape is regulated across heterotypic cells. We find that epithelial cells maintain the shape of neighboring sense-organ glia-neuron units in adult Caenorhabditis elegans (C. elegans). Hsp co-chaperone UNC-23/BAG2 prevents epithelial cell shape from deforming, and its loss causes head epithelia to stretch aberrantly during animal movement. In the sense-organ glia, amphid sheath (AMsh), this causes progressive fibroblast growth factor receptor (FGFR)-dependent disruption of the glial apical cytoskeleton. Resultant glial cell shape alteration causes concomitant shape change in glia-associated neuron endings. Epithelial UNC-23 maintenance of glia-neuron shape is specific both spatially, within a defined anatomical zone, and temporally, in a developmentally critical period. As all molecular components uncovered are broadly conserved across central and peripheral nervous systems, we posit that epithelia may similarly regulate glia-neuron architecture cross-species.

Mutations in the NXF-1:NXT-1 mRNA export complex affect gene-expression driven by the hsp-16.41 promoter.

The NXF-1 : NXT-1 heterodimer is essential for the nuclear export of mRNA. Here we describe three new alleles of nxf-1 and one allele of nxt-1 isolated from a forward genetic screen. These mutations cause no apparent phenotype under normal growth conditions, but partially suppress the lethality caused by heat-shock induced expression of the PEEL-1 toxin from P hsp-16.41 :: peel-1 . There is also decreased expression of P hsp-16.41 ::eGFP in an nxf-1 mutant. We propose that NXF-1 : NXT-1 influences the expression of heat-shock activated genes due to a role in the recruitment of the hsp-16.41 promoter to the nuclear pore complex during heat-shock.

Effectors of anterior morphogenesis in C. elegans embryos.

During embryogenesis the nascent Caenorhabditis elegans epidermis secretes an apical extracellular matrix (aECM) that serves as an external stabilizer, preventing deformation of the epidermis by mechanical forces exerted during morphogenesis. At present, the factors that contribute to aECM function are mostly unknown, including the aECM components themselves, their posttranslational regulators, and the pathways required for their secretion. Here we showed that two proteins previously linked to aECM function, SYM-3/FAM102A and SYM-4/WDR44, colocalize to intracellular and membrane-associated puncta and likely function in a complex. Proteomics experiments also suggested potential roles for SYM-3/FAM102A and SYM-4/WDR44 family proteins in intracellular trafficking. Nonetheless, we found no evidence to support a critical function for SYM-3 or SYM-4 in the apical deposition of two aECM components, NOAH-1 and FBN-1. Moreover, loss of a key splicing regulator of fbn-1, MEC-8/RBPMS2, had surprisingly little effect on the abundance or deposition of FBN-1. Using a focused screening approach, we identified 32 additional proteins that likely contribute to the structure and function of the embryonic aECM. We also characterized morphogenesis defects in embryos lacking mir-51 microRNA family members, which display a similar phenotype to mec-8; sym double mutants. Collectively, these findings add to our knowledge of factors controlling embryonic morphogenesis.

Pathways that affect anterior morphogenesis in C. elegans embryos.

During embryogenesis the nascent Caenorhabditis elegans epidermis secretes an apical extracellular matrix (aECM) that serves as an external stabilizer, preventing deformation of the epidermis by mechanical forces exerted during morphogenesis. We showed that two conserved proteins linked to this process, SYM-3/FAM102A and SYM-4/WDR44, colocalize to intracellular and membrane-associated puncta and likely function together in a complex. Proteomics data also suggested potential roles for FAM102A and WDR44 family proteins in intracellular trafficking, consistent with their localization patterns. Nonetheless, we found no evidence to support a clear function for SYM-3 or SYM-4 in the apical deposition of two aECM components, FBN-1 and NOAH. Surprisingly, loss of MEC-8/RBPMS2, a conserved splicing factor and regulator of fbn-1 , had little effect on the abundance or deposition of FBN-1 to the aECM. Using a focused screening approach, we identified 32 additional proteins that likely contribute to the structure and function of the embryonic aECM. Lastly, we examined morphogenesis defects in embryos lacking mir-51 microRNA family members, which display a related embryonic phenotype to mec-8; sym double mutants. Collectively, our findings add to our knowledge of pathways controlling embryonic morphogenesis. SUMMARY STATEMENT: We identify new proteins in apical ECM biology in C. elegans and provide evidence that SYM-3/FAM102A and SYM-4/WDR44 function together in trafficking but do not regulate apical ECM protein deposition.

G protein-coupled receptor kinase-2 (GRK-2) controls exploration through neuropeptide signaling in Caenorhabditis elegans.

Animals alter their behavior in manners that depend on environmental conditions as well as their developmental and metabolic states. For example, C. elegans is quiescent during larval molts or during conditions of satiety. By contrast, worms enter an exploration state when removed from food. Sensory perception influences movement quiescence (defined as a lack of body movement), as well as the expression of additional locomotor states in C. elegans that are associated with increased or reduced locomotion activity, such as roaming (exploration behavior) and dwelling (local search). Here we find that movement quiescence is enhanced, and exploration behavior is reduced in G protein-coupled receptor kinase grk-2 mutant animals. grk-2 was previously shown to act in chemosensation, locomotion, and egg-laying behaviors. Using neuron-specific rescuing experiments, we show that GRK-2 acts in multiple ciliated chemosensory neurons to control exploration behavior. grk-2 acts in opposite ways from the cGMP-dependent protein kinase gene egl-4 to control movement quiescence and exploration behavior. Analysis of mutants with defects in ciliated sensory neurons indicates that grk-2 and the cilium-structure mutants act in the same pathway to control exploration behavior. We find that GRK-2 controls exploration behavior in an opposite manner from the neuropeptide receptor NPR-1 and the neuropeptides FLP-1 and FLP-18. Finally, we show that secretion of the FLP-1 neuropeptide is negatively regulated by GRK-2 and that overexpression of FLP-1 reduces exploration behavior. These results define neurons and molecular pathways that modulate movement quiescence and exploration behavior.

Fixation and Immunostaining of Endogenous Proteins or Post-translational Modificationsin Caenorhabditis elegans.

Although the advent of genetically-encoded fluorescent markers, such as the green fluorescent protein (GFP; Chalfie et al., 1994 ), has enabled convenient visualization of gene expression in vivo, this method is generally not effective for detecting post-translational modifications because they are not translated from DNA sequences. Genetically-encoded, fluorescently-tagged transgene products can also be misleading for observing expression patterns because transgenes may lack endogenous regulatory DNA elements needed for precise regulation of expression that could result in over or under expression. Fluorescently-tagged proteins created by CRISPR genome editing are less prone to defective expression patterns because the loci retain endogenous DNA elements that regulate their transcription (Nance and Frøkjær-Jensen, 2019). However, even CRISPR alleles encoding heritable fluorescently-tagged protein markers can result in defects in function or localization of the gene product if the fluorescent tag obstructs or otherwise interferes with important protein interaction domains or affects the protein structure. Indirect immunofluorescence is a method for detecting endogenous gene expression or post-translational modifications without the need for transgenesis or genome editing. Here, we present a reliable protocol in which C. elegans nematodes are fixed, preserved, and permeabilized for staining with a primary antibody to bind proteins or post-translational modifications, which are then labeled with a secondary antibody conjugated to a fluorescent dye. Use of this method may be limited by the availability of (or ability to generate) a primary antibody that binds the epitope of interest in fixed animals. Thousands of animals are simultaneously subjected to a series of chemical treatments and washes in a single centrifuge tube, allowing large numbers of identically-treated stained animals to be examined. We have successfully used this protocol (O' Hagan et al., 2011 and 2017; Power et al., 2020 ) to preserve and detect post-translational modifications of tubulin in C. elegans ciliated sensory neurons and to detect non-modified endogenous protein (Topalidou and Chalfie, 2011).

Background mutation in strain RB2126 affects the locomotion behavior of flp-1 mutants.

The FLP-1 neuropeptide is involved in locomotion, mechanosensation, and reproduction. Strain RB2126 is reported to contain the flp-1(ok2811) mutation, a prospective null allele. Here we report that strain RB2126, in addition to flp-1(ok2811), also contains two additional background mutations. One of the mutations gives the animals a constitutive dauer phenotype (Daf-c), and complementation testing confirms that it is in the Daf-c gene daf-11. The second mutation is isolated based on the fact that it partially suppresses the elevated body curvature of flp-1(ok2811) mutants, but it remains uncloned. The updated genotype of RB2126 strain is flp-1(ok2811); daf-11(yak155); yak156.

Dopamine receptor DOP-1 engages a sleep pathway to modulate swimming in C. elegans.

Animals require robust yet flexible programs to support locomotion. Here we report a pathway that connects the D1-like dopamine receptor DOP-1 with a sleep mechanism to modulate swimming in C. elegans. We show that DOP-1 plays a negative role in sustaining swimming behavior. By contrast, a pathway through the D2-like dopamine receptor DOP-3 negatively regulates the initiation of swimming, but its impact fades quickly over a few minutes. We find that DOP-1 and the GPCR kinase (G-protein-coupled receptor kinase-2) function in the sleep interneuron RIS, where DOP-1 modulates the secretion of a sleep neuropeptide FLP-11. We further show that DOP-1 and FLP-11 act in the same pathway to modulate swimming. Together, these results delineate a functional connection between a dopamine receptor and a sleep program to regulate swimming in C. elegans. The temporal transition between DOP-3 and DOP-1 pathways highlights the dynamic nature of neuromodulation for rhythmic movements that persist over time.

EIPR1 controls dense-core vesicle cargo retention and EARP complex localization in insulin-secreting cells.

Dense-core vesicles (DCVs) are secretory vesicles found in neurons and endocrine cells. DCVs package and release cargoes including neuropeptides, biogenic amines, and peptide hormones. We recently identified the endosome-associated recycling protein (EARP) complex and the EARP-interacting-protein EIPR-1 as proteins important for controlling levels of DCV cargoes in Caenorhabditis elegans neurons. Here we determine the role of mammalian EIPR1 in insulinoma cells. We find that in Eipr1 KO cells, there is reduced insulin secretion, and mature DCV cargoes such as insulin and carboxypeptidase E (CPE) accumulate near the trans-Golgi network and are not retained in mature DCVs in the cell periphery. In addition, we find that EIPR1 is required for the stability of the EARP complex subunits and for the localization of EARP and its association with membranes, but EIPR1 does not affect localization or function of the related Golgi-associated retrograde protein (GARP) complex. EARP is localized to two distinct compartments related to its function: an endosomal compartment and a DCV biogenesis-related compartment. We propose that EIPR1 functions with EARP to control both endocytic recycling and DCV maturation.

Modulation of Gq-Rho Signaling by the ERK MAPK Pathway Controls Locomotion in Caenorhabditis elegans.

The heterotrimeric G protein Gq regulates neuronal activity through distinct downstream effector pathways. In addition to the canonical Gq effector phospholipase Cß, the small GTPase Rho was recently identified as a conserved effector of Gq. To identify additional molecules important for Gq signaling in neurons, we performed a forward genetic screen in the nematode Caenorhabditis elegans for suppressors of the hyperactivity and exaggerated waveform of an activated Gq mutant. We isolated two mutations affecting the MAP kinase scaffold protein KSR-1 and found that KSR-1 modulates locomotion downstream of, or in parallel to, the Gq-Rho pathway. Through epistasis experiments, we found that the core ERK MAPK cascade is required for Gq-Rho regulation of locomotion, but that the canonical ERK activator LET-60/Ras may not be required. Through neuron-specific rescue experiments, we found that the ERK pathway functions in head acetylcholine neurons to control Gq-dependent locomotion. Additionally, expression of activated LIN-45/Raf in head acetylcholine neurons is sufficient to cause an exaggerated waveform phenotype and hypersensitivity to the acetylcholinesterase inhibitor aldicarb, similar to an activated Gq mutant. Taken together, our results suggest that the ERK MAPK pathway modulates the output of Gq-Rho signaling to control locomotion behavior in C. elegans.

Dopamine negatively modulates the NCA ion channels in C. elegans.

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.

Caenorhabditis elegans HIF-1 Is Broadly Required for Survival in Hydrogen Sulfide.

Hydrogen sulfide is common in the environment, and is also endogenously produced by animal cells. Although hydrogen sulfide is often toxic, exposure to low levels of hydrogen sulfide improves outcomes in a variety of mammalian models of ischemia-reperfusion injury. In Caenorhabditis elegans, the initial transcriptional response to hydrogen sulfide depends on the hif-1 transcription factor, and hif-1 mutant animals die when exposed to hydrogen sulfide. In this study, we use rescue experiments to identify tissues in which hif-1 is required to survive exposure to hydrogen sulfide. We find that expression of hif-1 from the unc-14 promoter is sufficient to survive hydrogen sulfide. Although unc-14 is generally considered to be a pan-neuronal promoter, we show that it is active in many nonneuronal cells as well. Using other promoters, we show that pan-neuronal expression of hif-1 is not sufficient to survive exposure to hydrogen sulfide. Our data suggest that hif-1 is required in many different tissues to direct the essential response to hydrogen sulfide.

The dense-core vesicle maturation protein CCCP-1 binds RAB-2 and membranes through its C-terminal domain.

Dense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode Caenorhabditis elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here, we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. In addition, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.

The SEK-1 p38 MAP Kinase Pathway Modulates Gq Signaling in Caenorhabditis elegans.

Gq is a heterotrimeric G protein that is widely expressed in neurons and regulates neuronal activity. To identify pathways regulating neuronal Gq signaling, we performed a forward genetic screen in Caenorhabditis elegans for suppressors of activated Gq. One of the suppressors is an allele of sek-1, which encodes a mitogen-activated protein kinase kinase (MAPKK) in the p38 MAPK pathway. Here, we show that sek-1 mutants have a slow locomotion rate and that sek-1 acts in acetylcholine neurons to modulate both locomotion rate and Gq signaling. Furthermore, we find that sek-1 acts in mature neurons to modulate locomotion. Using genetic and behavioral approaches, we demonstrate that other components of the p38 MAPK pathway also play a positive role in modulating locomotion and Gq signaling. Finally, we find that mutants in the SEK-1 p38 MAPK pathway partially suppress an activated mutant of the sodium leak channel, NCA-1/NALCN, a downstream target of Gq signaling. Our results suggest that the SEK-1 p38 pathway may modulate the output of Gq signaling through NCA-1(unc-77).

The NCA-1 and NCA-2 Ion Channels Function Downstream of Gq and Rho To Regulate Locomotion in Caenorhabditis elegans.

The heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here, we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq-Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.

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.

Genetically separable functions of the MEC-17 tubulin acetyltransferase affect microtubule organization.

BACKGROUND: Microtubules (MTs) are formed from the lateral association of 11-16 protofilament chains of tubulin dimers, with most cells containing 13-protofilament (13-p) MTs. How these different MTs are formed is unknown, although the number of protofilaments may depend on the nature of the α- and ß-tubulins. RESULTS: Here we show that the enzymatic activity of the Caenorhabiditis elegans α-tubulin acetyltransferase (α-TAT) MEC-17 allows the production of 15-p MTs in the touch receptor neurons (TRNs) MTs. Without MEC-17, MTs with between 11 and 15 protofilaments are seen. Loss of this enzymatic activity also changes the number and organization of the TRN MTs and affects TRN axonal morphology. In contrast, enzymatically inactive MEC-17 is sufficient for touch sensitivity and proper process outgrowth without correcting the MT defects. Thus, in addition to demonstrating that MEC-17 is required for MT structure and organization, our results suggest that the large number of 15-p MTs, normally found in the TRNs, is not essential for mechanosensation. CONCLUSION: These experiments reveal a specific role for α-TAT in the formation of MTs and in the production of higher order MTs arrays. In addition, our results indicate that the α-TAT protein has functions that require acetyltransferase activity (such as the determination of protofilament number) and others that do not (presence of internal MT structures).

Crystal structure and RNA binding properties of the RNA recognition motif (RRM) and AlkB domains in human AlkB homolog 8 (ABH8), an enzyme catalyzing tRNA hypermodification.

Humans express nine paralogs of the bacterial DNA repair enzyme AlkB, an iron/2-oxoglutarate-dependent dioxygenase that reverses alkylation damage to nucleobases. The biochemical and physiological roles of these paralogs remain largely uncharacterized, hampering insight into the evolutionary expansion of the AlkB family. However, AlkB homolog 8 (ABH8), which contains RNA recognition motif (RRM) and methyltransferase domains flanking its AlkB domain, recently was demonstrated to hypermodify the anticodon loops in some tRNAs. To deepen understanding of this activity, we performed physiological and biophysical studies of ABH8. Using GFP fusions, we demonstrate that expression of the Caenorhabditis elegans ABH8 ortholog is widespread in larvae but restricted to a small number of neurons in adults, suggesting that its function becomes more specialized during development. In vitro RNA binding studies on several human ABH8 constructs indicate that binding affinity is enhanced by a basic α-helix at the N terminus of the RRM domain. The 3.0-Å-resolution crystal structure of a construct comprising the RRM and AlkB domains shows disordered loops flanking the active site in the AlkB domain and a unique structural Zn(II)-binding site at its C terminus. Although the catalytic iron center is exposed to solvent, the 2-oxoglutarate co-substrate likely adopts an inactive conformation in the absence of tRNA substrate, which probably inhibits uncoupled free radical generation. A conformational change in the active site coupled to a disorder-to-order transition in the flanking protein segments likely controls ABH8 catalytic activity and tRNA binding specificity. These results provide insight into the functional and structural adaptations underlying evolutionary diversification of AlkB domains.