Meisosomes, folded membrane microdomains between the apical extracellular matrix and epidermis.

Apical extracellular matrices (aECMs) form a physical barrier to the environment. In Caenorhabditis elegans, the epidermal aECM, the cuticle, is composed mainly of different types of collagen, associated in circumferential ridges separated by furrows. Here, we show that in mutants lacking furrows, the normal intimate connection between the epidermis and the cuticle is lost, specifically at the lateral epidermis, where, in contrast to the dorsal and ventral epidermis, there are no hemidesmosomes. At the ultrastructural level, there is a profound alteration of structures that we term 'meisosomes,' in reference to eisosomes in yeast. We show that meisosomes are composed of stacked parallel folds of the epidermal plasma membrane, alternately filled with cuticle. We propose that just as hemidesmosomes connect the dorsal and ventral epidermis, above the muscles, to the cuticle, meisosomes connect the lateral epidermis to it. Moreover, furrow mutants present marked modifications of the biomechanical properties of their skin and exhibit a constitutive damage response in the epidermis. As meisosomes co-localise to macrodomains enriched in phosphatidylinositol (4,5) bisphosphate, they could conceivably act, like eisosomes, as signalling platforms, to relay tensile information from the aECM to the underlying epidermis, as part of an integrated stress response to damage.

Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

ifas-1 is upregulated by fungal infection in a GPA-12 and STA-2-independent manner in the Caenorhabditis elegans epidermis.

Skin infection with the fungus Drechmeria coniospora leads to a transcriptional response in the worm epidermis. This involves an increased expression of a group of antimicrobial peptide (AMP) genes including those in the nlp-29 and cnc-2 clusters. The major pathways leading to the expression of these AMP genes have been well characterized and converge on the STAT transcription factor STA-2. We reported previously that expression in the epidermis of a constitutively active (gain of function, gf) form of the Gα protein GPA-12 (GPA-12gf) recapitulates much of the response to infection. To reveal parallel pathways activated by infection, we focus here on an effector gene that is not induced by GPA-12gf. This gene, ifas-1, encodes a protein with a fascin domain, associated with actin binding. Its induction upon fungal infection does not require sta-2. A transcriptional reporter revealed induction in the epidermis of ifas-1 by infection and wounding. Thus, ifas-1 represents part of a previously unexplored aspect of the innate immune response to infection.

unc-119 mutants have an increased fungal spore adhesion that is not rescued by Cb-unc-119.

If the cuticle acts as a protective barrier against environmental insults, several pathogens have developed strategies that use it as a way to infect C. elegans. The fungus Drechmeria coniospora produces spores that attach to the cuticle, before hyphae invade the body. Mutants with an altered surface coat, the outermost layer of the cuticle, including bus-2, bus-4, bus-12 and bus-17 show increased adhesion of fungal spores (Rouger et al, 2014; Zugasti et al, 2016). We unexpectedly found that D. coniospora spores attach unusually densely around the mouth of unc-119 mutants. Interestingly, this phenotype is not rescued by the C. briggsae unc-119 construct that is conventionally used to rescue neuronal unc-119 phenotypes.

Comparison of lipidome profiles of Caenorhabditis elegans-results from an inter-laboratory ring trial.

INTRODUCTION: Lipidomic profiling allows 100s if not 1000s of lipids in a sample to be detected and quantified. Modern lipidomics techniques are ultra-sensitive assays that enable the discovery of novel biomarkers in a variety of fields and provide new insight in mechanistic investigations. Despite much progress in lipidomics, there remains, as for all high throughput "omics" strategies, the need to develop strategies to standardize and integrate quality control into studies in order to enhance robustness, reproducibility, and usability of studies within specific fields and beyond. OBJECTIVES: We aimed to understand how much results from lipid profiling in the model organism Caenorhabditis elegans are influenced by different culture conditions in different laboratories. METHODS: In this work we have undertaken an inter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans and daf-2(e1370) mutants lacking a functional insulin receptor. Sample were collected from worms grown in four separate laboratories under standardized growth conditions. We used an UPLC-UHR-ToF-MS system allowing chromatographic separation before MS analysis. RESULTS: We found common qualitative changes in several marker lipids in samples from the individual laboratories. On the other hand, even in this controlled experimental system, the exact fold-changes for each marker varied between laboratories. CONCLUSION: Our results thus reveal a serious limitation to the reproducibility of current lipid profiling experiments and reveal challenges to the integration of such data from different laboratories.

New Strains for Tissue-Specific RNAi Studies in Caenorhabditis elegans.

RNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1(ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1(ne219) and the reported germline only RNAi strain, rrf-1(pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1(ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

Non-Canonical Caspase Activity Antagonizes p38 MAPK Stress-Priming Function to Support Development.

Recent studies have revealed non-canonical activities of apoptotic caspases involving specific modulation of gene expression, such as limiting asymmetric divisions of stem-like cell types. Here we report that CED-3 caspase negatively regulates an epidermal p38 stress-responsive MAPK pathway to promote larval development in C. elegans. We show that PMK-1 (p38 MAPK) primes animals for encounters with hostile environments at the expense of retarding post-embryonic development. CED-3 counters this function by directly cleaving PMK-1 to promote development. Moreover, we found that CED-3 and PMK-1 oppose each other to balance developmental and stress-responsive gene expression programs. Specifically, expression of more than 300 genes is inversely regulated by CED-3 and PMK-1. Analyses of these genes showed enrichment for epidermal stress-responsive factors, including the fatty acid synthase FASN-1, anti-microbial peptides, and genes involved in lethargus states. Our findings demonstrate a non-canonical role for a caspase in promoting development by limiting epidermal stress response programs.

Microtubule plus-end dynamics link wound repair to the innate immune response.

The skin protects animals from infection and physical damage. In Caenorhabditis elegans, wounding the epidermis triggers an immune reaction and a repair response, but it is not clear how these are coordinated. Previous work implicated the microtubule cytoskeleton in the maintenance of epidermal integrity (Chuang et al., 2016). Here, by establishing a simple wounding system, we show that wounding provokes a reorganisation of plasma membrane subdomains. This is followed by recruitment of the microtubule plus end-binding protein EB1/EBP-2 around the wound and actin ring formation, dependent on ARP2/3 branched actin polymerisation. We show that microtubule dynamics are required for the recruitment and closure of the actin ring, and for the trafficking of the key signalling protein SLC6/SNF-12 toward the injury site. Without SNF-12 recruitment, there is an abrogation of the immune response. Our results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of innate immunity.

Evolutionary plasticity in the innate immune function of Akirin.

Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NFκB. Although nematodes lack NFκB, Akirin is also indispensable for the expression of defence genes in the epidermis of Caenorhabditis elegans following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in C. elegans Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in C. elegans, our results give insight into the molecular evolution of lineage-specific signalling pathways.

Modulatory upregulation of an insulin peptide gene by different pathogens in C. elegans.

When an animal is infected, its innate immune response needs to be tightly regulated across tissues and coordinated with other aspects of organismal physiology. Previous studies with Caenorhabditis elegans have demonstrated that insulin-like peptide genes are differentially expressed in response to different pathogens. They represent prime candidates for conveying signals between tissues upon infection. Here, we focused on one such gene, ins-11 and its potential role in mediating cross-tissue regulation of innate immune genes. While diverse bacterial intestinal infections can trigger the up-regulation of ins-11 in the intestine, we show that epidermal infection with the fungus Drechmeria coniospora triggers an upregulation of ins-11 in the epidermis. Using the Shigella virulence factor OpsF, a MAP kinase inhibitor, we found that in both cases, ins-11 expression is controlled cell autonomously by p38 MAPK, but via distinct transcription factors, STA-2/STAT in the epidermis and HLH-30/TFEB in the intestine. We established that ins-11, and the insulin signaling pathway more generally, are not involved in the regulation of antimicrobial peptide gene expression in the epidermis. The up-regulation of ins-11 in the epidermis does, however, affect intestinal gene expression in a complex manner, and has a deleterious effect on longevity. These results support a model in which insulin signaling, via ins-11, contributes to the coordination of the organismal response to infection, influencing the allocation of resources in an infected animal.

Coordinated inhibition of C/EBP by Tribbles in multiple tissues is essential for Caenorhabditis elegans development.

BACKGROUND: Tribbles proteins are conserved pseudokinases that function to control kinase signalling and transcription in diverse biological processes. Abnormal function in human Tribbles has been implicated in a number of diseases including leukaemia, metabolic syndromes and cardiovascular diseases. Caenorhabditis elegans Tribbles NIPI-3 was previously shown to activate host defense upon infection by promoting the conserved PMK-1/p38 mitogen-activated protein kinase (MAPK) signalling pathway. Despite the prominent role of Tribbles proteins in many species, our knowledge of their mechanism of action is fragmented, and the in vivo functional relevance of their interactions with other proteins remains largely unknown. RESULTS: Here, by characterizing nipi-3 null mutants, we show that nipi-3 is essential for larval development and viability. Through analyses of genetic suppressors of nipi-3 null mutant lethality, we show that NIPI-3 negatively controls PMK-1/p38 signalling via transcriptional repression of the C/EBP transcription factor CEBP-1. We identified CEBP-1's transcriptional targets by ChIP-seq analyses and found them to be enriched in genes involved in development and stress responses. Unlike its cell-autonomous role in innate immunity, NIPI-3 is required in multiple tissues to control organismal development. CONCLUSIONS: Together, our data uncover an unprecedented crosstalk involving multiple tissues, in which NIPI-3 acts as a master regulator to inhibit CEBP-1 and the PMK-1/p38 MAPK pathway. In doing so, it keeps innate immunity in check and ensures proper organismal development.

A quantitative genome-wide RNAi screen in C. elegans for antifungal innate immunity genes.

BACKGROUND: Caenorhabditis elegans has emerged over the last decade as a useful model for the study of innate immunity. Its infection with the pathogenic fungus Drechmeria coniospora leads to the rapid up-regulation in the epidermis of genes encoding antimicrobial peptides. The molecular basis of antimicrobial peptide gene regulation has been previously characterized through forward genetic screens. Reverse genetics, based on RNAi, provide a complementary approach to dissect the worm's immune defenses. RESULTS: We report here the full results of a quantitative whole-genome RNAi screen in C. elegans for genes involved in regulating antimicrobial peptide gene expression. The results will be a valuable resource for those contemplating similar RNAi-based screens and also reveal the limitations of such an approach. We present several strategies, including a comprehensive class clustering method, to overcome these limitations and which allowed us to characterize the different steps of the interaction between C. elegans and the fungus D. coniospora, leading to a complete description of the MAPK pathway central to innate immunity in C. elegans. The results further revealed a cross-tissue signaling, triggered by mitochondrial dysfunction in the intestine, that suppresses antimicrobial peptide gene expression in the nematode epidermis. CONCLUSIONS: Overall, our results provide an unprecedented system's level insight into the regulation of C. elegans innate immunity. They represent a significant contribution to our understanding of host defenses and will lead to a better comprehension of the function and evolution of animal innate immunity.

The human PDZome: a gateway to PSD95-Disc large-zonula occludens (PDZ)-mediated functions.

Protein-protein interactions organize the localization, clustering, signal transduction, and degradation of cellular proteins and are therefore implicated in numerous biological functions. These interactions are mediated by specialized domains able to bind to modified or unmodified peptides present in binding partners. Among the most broadly distributed protein interaction domains, PSD95-disc large-zonula occludens (PDZ) domains are usually able to bind carboxy-terminal sequences of their partners. In an effort to accelerate the discovery of PDZ domain interactions, we have constructed an array displaying 96% of the human PDZ domains that is amenable to rapid two-hybrid screens in yeast. We have demonstrated that this array can efficiently identify interactions using carboxy-terminal sequences of PDZ domain binders such as the E6 oncoviral protein and protein kinases (PDGFRß, BRSK2, PCTK1, ACVR2B, and HER4); this has been validated via mass spectrometry analysis. Taking advantage of this array, we show that PDZ domains of Scrib and SNX27 bind to the carboxy-terminal region of the planar cell polarity receptor Vangl2. We also have demonstrated the requirement of Scrib for the promigratory function of Vangl2 and described the morphogenetic function of SNX27 in the early Xenopus embryo. The resource presented here is thus adapted for the screen of PDZ interactors and, furthermore, should facilitate the understanding of PDZ-mediated functions.

The pseudokinase NIPI-4 is a novel regulator of antimicrobial peptide gene expression.

Hosts have developed diverse mechanisms to counter the pathogens they face in their natural environment. Throughout the plant and animal kingdoms, the up-regulation of antimicrobial peptides is a common response to infection. In C. elegans, infection with the natural pathogen Drechmeria coniospora leads to rapid induction of antimicrobial peptide gene expression in the epidermis. Through a large genetic screen we have isolated many new mutants that are incapable of upregulating the antimicrobial peptide nlp-29 in response to infection (i.e. with a Nipi or 'no induction of peptide after infection' phenotype). More than half of the newly isolated Nipi mutants do not correspond to genes previously associated with the regulation of antimicrobial peptides. One of these, nipi-4, encodes a member of a nematode-specific kinase family. NIPI-4 is predicted to be catalytically inactive, thus to be a pseudokinase. It acts in the epidermis downstream of the PKC∂ TPA-1, as a positive regulator of nlp antimicrobial peptide gene expression after infection. It also controls the constitutive expression of antimicrobial peptide genes of the cnc family that are targets of TGFß regulation. Our results open the way for a more detailed understanding of how host defense pathways can be molded by environmental pathogens.

Unusual regulation of a STAT protein by an SLC6 family transporter in C. elegans epidermal innate immunity.

The cuticle and epidermis of Caenorhabditis elegans provide the first line of defense against invading pathogens. Upon invasion by the fungal pathogen Drechmeria coniospora, C. elegans responds by upregulating the expression of antimicrobial peptides (AMPs) in the epidermis via activation of at least two pathways, a neuroendocrine TGF-ß pathway and a p38 MAPK pathway. Here, we identify the sodium-neurotransmitter symporter SNF-12, a member of the solute carrier family (SLC6), as being essential for both these immune signaling pathways. We also identify the STAT transcription factor-like protein STA-2 as a direct physical interactor of SNF-12 and show that the two proteins function together to regulate AMP gene expression in the epidermis. Both SNF-12 and STA-2 act cell autonomously and specifically in the epidermis to govern the transcriptional response to fungal infection. These findings reveal an unorthodox mode of regulation for a STAT factor and highlight the molecular plasticity of innate immune signaling.

A genome-wide study of PDZ-domain interactions in C. elegans reveals a high frequency of non-canonical binding.

BACKGROUND: Proteins may evolve through the recruitment and modification of discrete domains, and in many cases, protein action can be dissected at the domain level. PDZ domains are found in many important structural and signaling complexes, and are generally thought to interact with their protein partners through a C-terminal consensus sequence. We undertook a comprehensive search for protein partners of all individual PDZ domains in C. elegans to characterize their function and mode of interaction. RESULTS: Coupling high-throughput yeast two-hybrid screens with extensive validation by co-affinity purification, we defined a domain-orientated interactome map. This integrates PDZ domain proteins in numerous cell-signaling pathways and shows that PDZ domain proteins are implicated in an unexpectedly wide range of cellular processes. Importantly, we uncovered a high frequency of non-canonical interactions, not involving the C-terminus of the protein partner, which were directly confirmed in most cases. We completed our study with the generation of a yeast array representing the entire set of PDZ domains from C. elegans and provide a proof-of-principle for its application to the discovery of PDZ domain targets for any protein or peptide of interest. CONCLUSIONS: We provide an extensive domain-centered dataset, together with a clone resource, that will help future functional study of PDZ domains. Through this unbiased approach, we revealed frequent non-canonical interactions between PDZ domains and their protein partners that will require a re-evaluation of this domain's molecular function.[The protein interactions from this publication have been submitted to the IMEx (http://www.imexconsortium.org) consortium through IntAct (PMID: 19850723) and assigned the identifier IM-14654].