Single-tissue proteomics in Caenorhabditis elegans reveals proteins resident in intestinal lysosome-related organelles.

The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry (MS)-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single-tissue MS-based proteomic analysis in small organisms and in its future utility.

Sensory integration of food availability and population density during the diapause exit decision involves insulin-like signaling in Caenorhabditis elegans.

Decisions made over long time scales, such as life cycle decisions, require coordinated interplay between sensory perception and sustained gene expression. The Caenorhabditis elegans dauer (or diapause) exit developmental decision requires sensory integration of population density and food availability to induce an all-or-nothing organismal-wide response, but the mechanism by which this occurs remains unknown. Here, we demonstrate how the ASJ chemosensory neurons, known to be critical for dauer exit, perform sensory integration at both the levels of gene expression and calcium activity. In response to favorable conditions, dauers rapidly produce and secrete the dauer exit-promoting insulin-like peptide INS-6. Expression of ins-6 in the ASJ neurons integrate population density and food level and can reflect decision commitment since dauers committed to exiting have higher ins-6 expression levels than those of non-committed dauers. Calcium imaging in dauers reveals that the ASJ neurons are activated by food, and this activity is suppressed by pheromone, indicating that sensory integration also occurs at the level of calcium transients. We find that ins-6 expression in the ASJ neurons depends on neuronal activity in the ASJs, cGMP signaling, a CaM-kinase pathway, and the pheromone components ascr#8 and ascr#2. We propose a model in which decision commitment to exit the dauer state involves an autoregulatory feedback loop in the ASJ neurons that promotes high INS-6 production and secretion. These results collectively demonstrate how insulin-like peptide signaling helps animals compute long-term decisions by bridging sensory perception to decision execution.

LINKIN-associated proteins necessary for tissue integrity during collective cell migration.

Cell adhesion plays essential roles in almost every aspect of metazoan biology. LINKIN (Human: ITFG1, Caenorhabditis elegans: lnkn-1) is a conserved transmembrane protein that has been identified to be necessary for tissue integrity during migration. In C. elegans, loss of lnkn-1 results in the detachment of the lead migratory cell from the rest of the developing male gonad. Previously, three interactors of ITFG1/lnkn-1 - RUVBL1/ruvb-1, RUVBL2/ruvb-2, and alpha-tubulin - were identified by immunoprecipitation-mass spectrometry (IP-MS) analysis using human HEK293T cells and then validated in the nematode male gonad. The ITFG1-RUVBL1 interaction has since been independently validated in a breast cancer cell line model that also implicates the involvement of the pair in metastasis. Here, we showed that epitope-tagged ITFG1 localized to the cell surface of MDA-MB-231 breast cancer cells. Using IP-MS analysis, we identified a new list of potential interactors of ITFG1. Loss-of-function analysis of their C. elegans orthologs found that three of the interactors - ATP9A/tat-5, NME1/ndk-1, and ANAPC2/apc-2 - displayed migratory detachment phenotypes similar to that of lnkn-1. Taken together with the other genes whose reduction-of-function phenotype is similar to that of lnkn-1 (notably cohesion and condensin), suggests the involvement of membrane remodeling and chromosome biology in LINKIN-dependent cell adhesion and supports the hypothesis for a structural role of chromosomes in post-mitotic cells.

Body stiffness is a mechanical property that facilitates contact-mediated mate recognition in Caenorhabditis elegans.

Physical contact is prevalent in the animal kingdom to recognize suitable mates by decoding information about sex, species, and maturity. Although chemical cues for mate recognition have been extensively studied, the role of mechanical cues remains elusive. Here, we show that C. elegans males recognize conspecific and reproductive mates through short-range cues, and that the attractiveness of potential mates depends on the sex and developmental stages of the hypodermis. We find that a particular group of cuticular collagens is required for mate attractiveness. These collagens maintain body stiffness to sustain mate attractiveness but do not affect the surface properties that evoke the initial step of mate recognition, suggesting that males utilize multiple sensory mechanisms to recognize suitable mates. Manipulations of body stiffness via physical interventions, chemical treatments, and 3D-printed bionic worms indicate that body stiffness is a mechanical property for mate recognition and increases mating efficiency. Our study thus extends the repertoire of sensory cues of mate recognition in C. elegans and provides a paradigm to study the important roles of mechanosensory cues in social behaviors.

Loss of famh-136/ FAM136A results in minor locomotion and behavioral changes in Caenorhabditis elegans.

Not much is known about FAM136A, a human gene that may be involved in Meniere's disease and is conserved throughout animals. To understand the function of famh-136 , the Caenorhabditis elegans ortholog of FAM136A, loss of function alleles of the gene were generated. We find that loss of famh-136 function results in minor but significant changes to the locomotion and behavior.

Brain-wide bidirectional neuropeptide modulation of individual neuron classes regulates a developmental decision.

Secreted neuromodulators, like biogenic amines and neuropeptides, can reconfigure circuit functions both locally and at a distance and establish global brain states that alter circuit outputs over prolonged timescales.1-3 Despite their diversity and ubiquitous presence, many studies on neuromodulation tend to focus on dissecting the function and site of action of individual neuropeptides. Here, we take a different approach by conducting a systems-level investigation of neuropeptide receptor signaling function and cell-type-specific distribution in the context of the Caenorhabditis elegans diapause entry developmental decision. C. elegans diapause entry is controlled by sensory perception of external factors and is regulated by neuropeptide signaling.4-8 We performed a comprehensive functional screen of neuropeptide receptor mutants for pheromone-induced diapause entry phenotypes and integrated these results with published C. elegans single-cell RNA-seq data to reveal that almost all neuron classes expressed at least one receptor with a role in diapause entry.9 Our receptor expression analysis also identified four highly modulated neural hubs with no previously reported roles in diapause entry that are distributed throughout the animal's body, possibly as a means of synchronizing the whole-organism transition into the appropriate larval morph. Furthermore, most neuron classes expressed unique neuropeptide receptor repertoires that have opposing effects on the diapause entry decision. We propose that brain-wide antagonistic neuropeptide modulation of individual neuron classes by distinct neuropeptide receptor subsets could serve as a strategy against overmodulation and that this motif might generalize to other decision-making paradigms in other organisms.

Interneuron control of C. elegans developmental decision-making.

Natural environments are highly dynamic, and this complexity challenges animals to accurately integrate external cues to shape their responses. Adaptive developmental plasticity enables organisms to remodel their physiology, morphology, and behavior to better suit the predicted future environment and ultimately enhance their ecological success.1 Understanding how an animal generates a neural representation of current and forecasted environmental conditions and converts these circuit computations into a predictive adaptive physiological response may provide fundamental insights into the molecular and cellular basis of decision-making over developmentally relevant timescales. Although it is known that sensory cues usually trigger the developmental switch and that downstream inter-tissue signaling pathways enact the alternative developmental phenotype, the integrative neural mechanisms that transduce external inputs into effector pathways are less clear.2,3 In adverse environments, Caenorhabditis elegans larvae can enter a stress-resistant diapause state with arrested metabolism and reproductive physiology.4 Amphid sensory neurons feed into both rapid chemotactic and short-term foraging mode decisions, mediated by amphid and pre-motor interneurons, as well as the long-term diapause entry decision. Here, we identify amphid interneurons that integrate pheromone cues and propagate this information via a neuropeptidergic pathway to influence larval developmental fate, bypassing the pre-motor system. AIA interneuron-derived FLP-2 neuropeptide signaling promotes reproductive growth, and AIA activity is suppressed by pheromones. FLP-2 signaling is inhibited by upstream glutamatergic transmission via the metabotropic receptor MGL-1 and mediated by the broadly expressed neuropeptide G-protein-coupled receptor NPR-30. Thus, metabotropic signaling allows the reuse of parts of a sensory system for a decision with a distinct timescale.

A conserved behavioral role for a nematode interneuron neuropeptide receptor.

Neuropeptides are evolutionarily conserved modulators of many aspects of animal behavior and physiology, and expand the repertoire of processes that can be controlled by a limited number of neurons. Deciphering the neuropeptidergic codes that govern distinct processes requires systematic functional analyses of neuropeptides and their cognate receptors. Even in well-studied model organisms like Caenorhabditis elegans, however, such efforts have been precluded by a lack of mutant reagents. Here, we generated and screened 21 C. elegans neuropeptide G-protein coupled receptor mutants with no pre-existing reagents for the touch-evoked escape response, and implicated six receptors expressed in diverse neuron classes representing multiple circuit levels in this behavior. We further characterized the mutant with the most severe phenotype, frpr-14, which was defective in multiple behavioral paradigms. We leveraged this range of phenotypes to reveal that FRPR-14 modulation of different precommand interneuron classes, AVH and AIB, can drive distinct behavioral subsets, demonstrating cellular context-dependent roles for FRPR-14 signaling. We then show that Caenorhabditis briggsae CBR-FRPR-14 modulates an AVH-like interneuron pair to regulate the same behaviors as C. elegans but to a smaller extent. Our results also suggest that differences in touch-evoked escape circuit architecture between closely related species results from changes in neuropeptide receptor expression pattern, as opposed to ligand-receptor pairing. This study provides insights into the principles utilized by a compact, multiplexed nervous system to generate intraspecific behavioral complexity and interspecific variation.

Signaling by AWC Olfactory Neurons Is Necessary for Caenorhabditis elegans' Response to Prenol, an Odor Associated with Nematode-Infected Insects.

Chemosensation plays a role in the behaviors and life cycles of numerous organisms, including nematodes. Many guilds of nematodes exist, ranging from the free-living Caenorhabditis elegans to various parasitic species such as entomopathogenic nematodes (EPNs), which are parasites of insects. Despite ecological differences, previous research has shown that both EPNs and C. elegans respond to prenol (3-methyl-2-buten-1-ol), an odor associated with EPN infections. However, it is unclear how C. elegans responds to prenol. By utilizing natural variation and genetic neuron ablation to investigate the response of C. elegans to prenol, we found that the AWC neurons are involved in the detection of prenol and that several genes (including dcap-1, dcap-2, and clec-39) influence response to this odorant. Furthermore, we identified that the response to prenol is mediated by the canonically proposed pathway required for other AWC-sensed attractants. However, upon testing genetically diverse isolates, we found that the response of some strains to prenol differed from their response to isoamyl alcohol, suggesting that the pathways mediating response to these two odorants may be genetically distinct. Further, evaluations leveraging natural variation and genome wide association revealed specific genes that influence nematode behavior and provide a foundation for future studies to better understand the role of prenol in nematode behavioral ecology.

Modular metabolite assembly in Caenorhabditis elegans depends on carboxylesterases and formation of lysosome-related organelles.

Signaling molecules derived from attachment of diverse metabolic building blocks to ascarosides play a central role in the life history of C. elegans and other nematodes; however, many aspects of their biogenesis remain unclear. Using comparative metabolomics, we show that a pathway mediating formation of intestinal lysosome-related organelles (LROs) is required for biosynthesis of most modular ascarosides as well as previously undescribed modular glucosides. Similar to modular ascarosides, the modular glucosides are derived from highly selective assembly of moieties from nucleoside, amino acid, neurotransmitter, and lipid metabolism, suggesting that modular glucosides, like the ascarosides, may serve signaling functions. We further show that carboxylesterases that localize to intestinal organelles are required for the assembly of both modular ascarosides and glucosides via ester and amide linkages. Further exploration of LRO function and carboxylesterase homologs in C. elegans and other animals may reveal additional new compound families and signaling paradigms.

An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9.

Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2 We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.