Mono-UFMylation promotes misfolding-associated secretion of α-synuclein.

Stressed cells secret misfolded proteins lacking signaling sequence via an unconventional protein secretion (UcPS) pathway, but how misfolded proteins are targeted selectively in UcPS is unclear. Here, we report that misfolded UcPS clients are subject to modification by a ubiquitin-like protein named ubiquitin-fold modifier 1 (UFM1). Using α-synuclein (α-Syn) as a UcPS model, we show that mutating the UFMylation sites in α-Syn or genetic inhibition of the UFMylation system mitigates α-Syn secretion, whereas overexpression of UFBP1, a component of the endoplasmic reticulum-associated UFMylation ligase complex, augments α-Syn secretion in mammalian cells and in model organisms. UFM1 itself is cosecreted with α-Syn, and the serum UFM1 level correlates with that of α-Syn. Because UFM1 can be directly recognized by ubiquitin specific peptidase 19 (USP19), a previously established UcPS stimulator known to associate with several chaperoning activities, UFMylation might facilitate substrate engagement by USP19, allowing stringent and regulated selection of misfolded proteins for secretion and proteotoxic stress alleviation.

O-GlcNAc transferase plays a non-catalytic role in C. elegans male fertility.

Animal behavior is influenced by the competing drives to maintain energy and to reproduce. The balance between these evolutionary pressures and how nutrient signaling pathways intersect with mating remains unclear. The nutrient sensor O-GlcNAc transferase, which post-translationally modifies intracellular proteins with a single monosaccharide, is responsive to cellular nutrient status and regulates diverse biological processes. Though essential in most metazoans, O-GlcNAc transferase (ogt-1) is dispensable in Caenorhabditis elegans, allowing genetic analysis of its physiological roles. Compared to control, ogt-1 males had a four-fold reduction in mean offspring, with nearly two thirds producing zero progeny. Interestingly, we found that ogt-1 males transferred sperm less often, and virgin males had reduced sperm count. ogt-1 males were also less likely to engage in mate-searching and mate-response behaviors. Surprisingly, we found normal fertility for males with hypodermal expression of ogt-1 and for ogt-1 strains with catalytic-dead mutations. This suggests OGT-1 serves a non-catalytic function in the hypodermis impacting male fertility and mating behavior. This study builds upon research on the nutrient sensor O-GlcNAc transferase and demonstrates a role it plays in the interplay between the evolutionary drives for reproduction and survival.

New Roles for the Heterochronic Transcription Factor LIN-29 in Cuticle Maintenance and Lipid Metabolism at the Larval-to-Adult Transition in Caenorhabditis elegans.

Temporal regulation of gene expression is a crucial aspect of metazoan development. In the roundworm Caenorhabditis elegans, the heterochronic pathway controls multiple developmental events in a time-specific manner. The most downstream effector of this pathway, the zinc-finger transcription factor LIN-29, acts in the last larval stage (L4) to regulate elements of the larval-to-adult switch. Here, we explore new LIN-29 targets and their implications for this developmental transition. We used RNA-sequencing to identify genes differentially expressed between animals misexpressing LIN-29 at an early time point and control animals. Among 230 LIN-29-activated genes, we found that genes encoding cuticle collagens were overrepresented. Interestingly, expression of lin-29 and some of these collagens was increased in adults with cuticle damage, suggesting a previously unknown function for LIN-29 in adult cuticle maintenance. On the other hand, genes involved in fat metabolism were enriched among 350 LIN-29-downregulated targets. We used mass spectrometry to assay lipid content in animals overexpressing LIN-29 and observed reduced fatty acid levels. Many LIN-29-repressed genes are normally expressed in the intestine, suggesting cell-nonautonomous regulation. We identified several LIN-29 upregulated genes encoding signaling molecules that may act as mediators in the regulation of intestinally expressed genes encoding fat metabolic enzymes and vitellogenins. Overall, our results support the model of LIN-29 as a major regulator of adult cuticle synthesis and integrity, and as the trigger for metabolic changes that take place at the important transition from rapid growth during larval life to slower growth and offspring production during adulthood.

Nutrient-Driven O-GlcNAcylation at Promoters Impacts Genome-Wide RNA Pol II Distribution.

Nutrient-driven O-GlcNAcylation has been linked to epigenetic regulation of gene expression in metazoans. In C. elegans, O-GlcNAc marks the promoters of over 800 developmental, metabolic, and stress-related genes; these O-GlcNAc marked genes show a strong 5', promoter-proximal bias in the distribution of RNA Polymerase II (Pol II). In response to starvation or feeding, the steady state distribution of O-GlcNAc at promoters remain nearly constant presumably due to dynamic cycling mediated by the transferase OGT-1 and the O-GlcNAcase OGA-1. However, in viable mutants lacking either of these enzymes of O-GlcNAc metabolism, the nutrient-responsive GlcNAcylation of promoters is dramatically altered. Blocked O-GlcNAc cycling leads to a striking nutrient-dependent accumulation of O-GlcNAc on RNA Pol II. O-GlcNAc cycling mutants also show an exaggerated, nutrient-responsive redistribution of promoter-proximal RNA Pol II isoforms and extensive transcriptional deregulation. Our findings suggest a complex interplay between the O-GlcNAc modification at promoters, the kinase-dependent "CTD-code," and co-factors regulating RNA Pol II dynamics. Nutrient-responsive O-GlcNAc cycling may buffer the transcriptional apparatus from dramatic swings in nutrient availability by modulating promoter activity to meet metabolic and developmental needs.

A Genetic Analysis of the Caenorhabditis elegans Detoxification Response.

Oxidative damage contributes to human diseases of aging including diabetes, cancer, and cardiovascular disorders. Reactive oxygen species resulting from xenobiotic and endogenous metabolites are sensed by a poorly understood process, triggering a cascade of regulatory factors and leading to the activation of the transcription factor Nrf2 (Nuclear factor-erythroid-related factor 2, SKN-1 in Caenorhabditis elegans). Nrf2/SKN-1 activation promotes the induction of the phase II detoxification system that serves to limit oxidative stress. We have extended a previous C. elegans genetic approach to explore the mechanisms by which a phase II enzyme is induced by endogenous and exogenous oxidants. The xrep (xenobiotics response pathway) mutants were isolated as defective in their ability to properly regulate the induction of a glutathione S-transferase (GST) reporter. The xrep-1 gene was previously identified as wdr-23, which encodes a C. elegans homolog of the mammalian ß-propeller repeat-containing protein WDR-23 Here, we identify and confirm the mutations in xrep-2, xrep-3, and xrep-4 The xrep-2 gene is alh-6, an ortholog of a human gene mutated in familial hyperprolinemia. The xrep-3 mutation is a gain-of-function allele of skn-1 The xrep-4 gene is F46F11.6, which encodes a F-box-containing protein. We demonstrate that xrep-4 alters the stability of WDR-23 (xrep-1), a key regulator of SKN-1 (xrep-3). Epistatic relationships among the xrep mutants and their interacting partners allow us to propose an ordered genetic pathway by which endogenous and exogenous stressors induce the phase II detoxification response.

Caenorhabditis elegans DAF-2 as a Model for Human Insulin Receptoropathies.

Human exome sequencing has dramatically increased the rate of identification of disease-associated polymorphisms. However, examining the functional consequences of those variants has created an analytic bottleneck. Insulin-like signaling in Caenorhabditis elegans has long provided a model to assess consequences of human insulin signaling mutations, but this has not been evaluated in the context of current genetic tools. We have exploited strains derived from the Million Mutation Project (MMP) and gene editing to explore further the evolutionary relationships and conservation between the human and C. elegans insulin receptors. Of 40 MMP alleles analyzed in the C. elegans insulin-like receptor gene DAF-2, 35 exhibited insulin-like signaling indistinguishable from wild-type animals, indicating tolerated mutations. Five MMP alleles proved to be novel dauer-enhancing mutations, including one new allele in the previously uncharacterized C-terminus of DAF-2 CRISPR-Cas9 genome editing was used to confirm the phenotypic consequence of six of these DAF-2 mutations and to replicate an allelic series of known human disease mutations in a highly conserved tyrosine kinase active site residue, demonstrating the utility of C. elegans for directly modeling human disease. Our results illustrate the challenges associated with prediction of the phenotypic consequences of amino acid substitutions, the value of assaying mutant isoform function in vivo, and how recently developed tools and resources afford the opportunity to expand our understanding even of highly conserved regulatory modules such as insulin signaling. This approach may prove generally useful for modeling phenotypic consequences of candidate human pathogenic mutations in conserved signaling and developmental pathways.

Regulation of UNC-130/FOXD-mediated mesodermal patterning in C. elegans.

Spatial polarity cues in animals are used repeatedly during development for many processes, including cell fate determination, cell migration, and axon guidance. In Caenorhabditis elegans, the body wall muscle extends the length of the animal in four distinct quadrants and generates an UNC-129/TGF-ß-related signal that is much higher in the dorsal two muscle quadrants compared to their ventral counterparts. This pattern of unc-129 expression requires the activity of the proposed transcriptional repressor UNC-130/FOXD whose body wall muscle activity is restricted to the ventral two body wall muscle quadrants. To understand how these dorsal-ventral differences in UNC-130 activity are established and maintained, we have analyzed the regulation of unc-130 expression and the distribution of UNC-130 protein. We have identified widespread, cis-acting elements in the unc-130 promoter that function to positively regulate ventral body wall muscle expression and negatively regulate dorsal body wall muscle expression. We have defined the temporal distribution of UNC-130 protein in body wall muscle cells during embryogenesis, demonstrated that this pattern is required to establish the dorsal-ventral polarity of UNC-129/TGF-ß, and shown that UNC-130 is not required post-embryonically to maintain the asymmetry of body wall muscle unc-129 expression. Finally, we have tested the impact of the depletion of a variety of transcription factors, repressors, and signaling molecules to identify additional regulators of body wall muscle UNC-130 polarity. Our results confirm and extend earlier studies to clarify the mechanisms by which UNC-130 is controlled and affects the pattern of unc-129 expression in body wall muscle. These results further our understanding of the transcriptional logic behind the generation of polarity cues involving this poorly understood subclass of Forkhead factors.

Myogenic conversion and transcriptional profiling of embryonic blastomeres in Caenorhabditis elegans.

Myogenesis has proven to be a powerful paradigm for understanding cell fate specification and differentiation in many model organisms. This includes the nematode Caenorhabditis elegans for which the genetic, cellular, and molecular tools have allowed an in-depth understanding of muscle development. One tool not yet available in C. elegans is a robust, pure and prolific cell culture system to study myogenesis. As an alternative, this chapter describes a method by which the cell fates of early, uncommitted blastomeres in the embryo are converted to a myogenic lineage. This technique permits the nearly synchronous induction of myogenesis in vivo with the potential to generate a nearly homogeneous population of cells. Coupled with the RNA isolation and cDNA amplification methods that are also described, one can now profile gene expression throughout myogenesis using any platform of choice (e.g. expression arrays, next generation sequencing). Although limited by the artificial nature of this developing mass of muscle inside the eggshell, blastomere conversion and transcriptional profiling is a very powerful tool to investigate changes in gene expression associated with myogenesis in C. elegans that is applicable to many different cell types. When coupled with next generation sequencing, the method has the potential to yield a very high-resolution map of changes in gene expression throughout myogenesis.

O-linked-N-acetylglucosamine cycling and insulin signaling are required for the glucose stress response in Caenorhabditis elegans.

In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. Here we demonstrate that O-GlcNAc cycling and insulin signaling are both essential components of the C. elegans response to glucose stress. A number of insulin-dependent processes were found to be sensitive to glucose stress, including fertility, reproductive timing, and dauer formation, yet each of these differed in their threshold of sensitivity to glucose excess. Our findings suggest that O-GlcNAc cycling and insulin signaling are both required for a robust and adaptable response to glucose stress, but these two pathways show complex and interdependent roles in the maintenance of glucose-insulin homeostasis.

Dynamic O-GlcNAc cycling at promoters of Caenorhabditis elegans genes regulating longevity, stress, and immunity.

Nutrient-driven O-GlcNAcylation of key components of the transcription machinery may epigenetically modulate gene expression in metazoans. The global effects of GlcNAcylation on transcription can be addressed directly in C. elegans because knockouts of the O-GlcNAc cycling enzymes are viable and fertile. Using anti-O-GlcNAc ChIP-on-chip whole-genome tiling arrays on wild-type and mutant strains, we detected over 800 promoters where O-GlcNAc cycling occurs, including microRNA loci and multigene operons. Intriguingly, O-GlcNAc-marked promoters are biased toward genes associated with PIP3 signaling, hexosamine biosynthesis, and lipid/carbohydrate metabolism. These marked genes are linked to insulin-like signaling, metabolism, aging, stress, and pathogen-response pathways in C. elegans. Whole-genome transcriptional profiling of the O-GlcNAc cycling mutants confirmed dramatic deregulation of genes in these key pathways. As predicted, the O-GlcNAc cycling mutants show altered lifespan and UV stress susceptibility phenotypes. We propose that O-GlcNAc cycling at promoters participates in a molecular program impacting nutrient-responsive pathways in C. elegans, including stress, pathogen response, and adult lifespan. The observed impact of O-GlcNAc cycling on both signaling and transcription in C. elegans has important implications for human diseases of aging, including diabetes and neurodegeneration.

A widespread distribution of genomic CeMyoD binding sites revealed and cross validated by ChIP-Chip and ChIP-Seq techniques.

Identifying transcription factor binding sites genome-wide using chromatin immunoprecipitation (ChIP)-based technology is becoming an increasingly important tool in addressing developmental questions. However, technical problems associated with factor abundance and suitable ChIP reagents are common obstacles to these studies in many biological systems. We have used two completely different, widely applicable methods to determine by ChIP the genome-wide binding sites of the master myogenic regulatory transcription factor HLH-1 (CeMyoD) in C. elegans embryos. The two approaches, ChIP-seq and ChIP-chip, yield strongly overlapping results revealing that HLH-1 preferentially binds to promoter regions of genes enriched for E-box sequences (CANNTG), known binding sites for this well-studied class of transcription factors. HLH-1 binding sites were enriched upstream of genes known to be expressed in muscle, consistent with its role as a direct transcriptional regulator. HLH-1 binding was also detected at numerous sites unassociated with muscle gene expression, as has been previously described for its mouse homolog MyoD. These binding sites may reflect several additional functions for HLH-1, including its interactions with one or more co-factors to activate (or repress) gene expression or a role in chromatin organization distinct from direct transcriptional regulation of target genes. Our results also provide a comparison of ChIP methodologies that can overcome limitations commonly encountered in these types of studies while highlighting the complications of assigning in vivo functions to identified target sites.

Loss of the transcriptional repressor PAG-3/Gfi-1 results in enhanced neurosecretion that is dependent on the dense-core vesicle membrane protein IDA-1/IA-2.

It is generally accepted that neuroendocrine cells regulate dense core vesicle (DCV) biogenesis and cargo packaging in response to secretory demands, although the molecular mechanisms of this process are poorly understood. One factor that has previously been implicated in DCV regulation is IA-2, a catalytically inactive protein phosphatase present in DCV membranes. Our ability to directly visualize a functional, GFP-tagged version of an IA-2 homolog in live Caenorhabditis elegans animals has allowed us to capitalize on the genetics of the system to screen for mutations that disrupt DCV regulation. We found that loss of activity in the transcription factor PAG-3/Gfi-1, which functions as a repressor in many systems, results in a dramatic up-regulation of IDA-1/IA-2 and other DCV proteins. The up-regulation of DCV components was accompanied by an increase in presynaptic DCV numbers and resulted in phenotypes consistent with increased neuroendocrine secretion. Double mutant combinations revealed that these PAG-3 mutant phenotypes were dependent on wild type IDA-1 function. Our results support a model in which IDA-1/IA-2 is a critical element in DCV regulation and reveal a novel genetic link to PAG-3-mediated transcriptional regulation. To our knowledge, this is the first mutation identified that results in increased neurosecretion, a phenotype that has clinical implications for DCV-mediated secretory disorders.

Caudal-like PAL-1 directly activates the bodywall muscle module regulator hlh-1 in C. elegans to initiate the embryonic muscle gene regulatory network.

Previous work in C. elegans has shown that posterior embryonic bodywall muscle lineages are regulated through a genetically defined transcriptional cascade that includes PAL-1/Caudal-mediated activation of muscle-specific transcription factors, including HLH-1/MRF and UNC-120/SRF, which together orchestrate specification and differentiation. Using chromatin immunoprecipitation (ChIP) in embryos, we now demonstrate direct binding of PAL-1 in vivo to an hlh-1 enhancer element. Through mutational analysis of the evolutionarily conserved sequences within this enhancer, we identify two cis-acting elements and their associated transacting factors (PAL-1 and HLH-1) that are crucial for the temporal-spatial expression of hlh-1 and proper myogenesis. Our data demonstrate that hlh-1 is indeed a direct target of PAL-1 in the posterior embryonic C. elegans muscle lineages, defining a novel in vivo binding site for this crucial developmental regulator. We find that the same enhancer element is also a target of HLH-1 positive auto regulation, underlying (at least in part) the sustained high levels of CeMyoD in bodywall muscle throughout development. Together, these results provide a molecular framework for the gene regulatory network activating the muscle module during embryogenesis.

ELT-2 is the predominant transcription factor controlling differentiation and function of the C. elegans intestine, from embryo to adult.

Starting with SAGE-libraries prepared from C. elegans FAC-sorted embryonic intestine cells (8E-16E cell stage), from total embryos and from purified oocytes, and taking advantage of the NextDB in situ hybridization data base, we define sets of genes highly expressed from the zygotic genome, and expressed either exclusively or preferentially in the embryonic intestine or in the intestine of newly hatched larvae; we had previously defined a similarly expressed set of genes from the adult intestine. We show that an extended TGATAA-like sequence is essentially the only candidate for a cis-acting regulatory motif common to intestine genes expressed at all stages. This sequence is a strong ELT-2 binding site and matches the sequence of GATA-like sites found to be important for the expression of every intestinal gene so far analyzed experimentally. We show that the majority of these three sets of highly expressed intestinal-specific/intestinal-enriched genes respond strongly to ectopic expression of ELT-2 within the embryo. By flow-sorting elt-2(null) larvae from elt-2(+) larvae and then preparing Solexa/Illumina-SAGE libraries, we show that the majority of these genes also respond strongly to loss-of-function of ELT-2. To test the consequences of loss of other transcription factors identified in the embryonic intestine, we develop a strain of worms that is RNAi-sensitive only in the intestine; however, we are unable (with one possible exception) to identify any other transcription factor whose intestinal loss-of-function causes a phenotype of comparable severity to the phenotype caused by loss of ELT-2. Overall, our results support a model in which ELT-2 is the predominant transcription factor in the post-specification C. elegans intestine and participates directly in the transcriptional regulation of the majority (>80%) of intestinal genes. We present evidence that ELT-2 plays a central role in most aspects of C. elegans intestinal physiology: establishing the structure of the enterocyte, regulating enzymes and transporters involved in digestion and nutrition, responding to environmental toxins and pathogenic infections, and regulating the downstream intestinal components of the daf-2/daf-16 pathway influencing aging and longevity.

The embryonic muscle transcriptome of Caenorhabditis elegans.

BACKGROUND: The force generating mechanism of muscle is evolutionarily ancient; the fundamental structural and functional components of the sarcomere are common to motile animals throughout phylogeny. Recent evidence suggests that the transcription factors that regulate muscle development are also conserved. Thus, a comprehensive description of muscle gene expression in a simple model organism should define a basic muscle transcriptome that is also found in animals with more complex body plans. To this end, we applied microarray profiling of Caenorhabtidis elegans cells (MAPCeL) to muscle cell populations extracted from developing C. elegans embryos. RESULTS: We used fluorescence-activated cell sorting to isolate myo-3::green fluorescent protein (GFP) positive muscle cells, and their cultured derivatives, from dissociated early C. elegans embryos. Microarray analysis identified 7,070 expressed genes, 1,312 of which are enriched in the myo-3::GFP positive cell population relative to the average embryonic cell. The muscle enriched gene set was validated by comparisons with known muscle markers, independently derived expression data, and GFP reporters in transgenic strains. These results confirm the utility of MAPCeL for cell type specific expression profiling and reveal that 60% of these transcripts have human homologs. CONCLUSION: This study provides a comprehensive description of gene expression in developing C. elegans embryonic muscle cells. The finding that more than half of these muscle enriched transcripts encode proteins with human homologs suggests that mutant analysis of these genes in C. elegans could reveal evolutionarily conserved models of muscle gene function, with ready application to human muscle pathologies.

Defining the transcriptional redundancy of early bodywall muscle development in C. elegans: evidence for a unified theory of animal muscle development.

Myogenic regulatory factors (MRFs) are required for mammalian skeletal myogenesis. In contrast, bodywall muscle is readily detectable in Caenorhabditis elegans embryos lacking activity of the lone MRF ortholog HLH-1, indicating that additional myogenic factors must function in the nematode. We find that two additional C. elegans proteins, UNC-120/SRF and HND-1/HAND, can convert naïve blastomeres to muscle when overproduced ectopically in the embryo. In addition, we have used genetic null mutants to demonstrate that both of these factors act in concert with HLH-1 to regulate myogenesis. Loss of all three factors results in embryos that lack detectable bodywall muscle differentiation, identifying this trio as a set that is both necessary and sufficient for bodywall myogenesis in C. elegans. In mammals, SRF and HAND play prominent roles in regulating smooth and cardiac muscle development. That C. elegans bodywall muscle development is dependent on transcription factors that are associated with all three types of mammalian muscle supports a theory that all animal muscle types are derived from a common ancestral contractile cell type.

The myogenic potency of HLH-1 reveals wide-spread developmental plasticity in early C. elegans embryos.

In vertebrates, striated muscle development depends on both the expression of members of the myogenic regulatory factor family (MRFs) and on extrinsic cellular cues, including Wnt signaling. The 81 embryonically born body wall muscle cells in C. elegans are comparable to the striated muscle of vertebrates. These muscle cells all express the gene hlh-1, encoding HLH-1 (CeMyoD) which is the only MRF-related factor in the nematode. However, genetic studies have shown that body wall muscle development occurs in the absence of HLH-1 activity, making the role of this factor in nematode myogenesis unclear. By ectopically expressing hlh-1 in early blastomeres of the C. elegans embryo, we show that CeMyoD is a bona fide MRF that can convert almost all cells to a muscle-like fate, regardless of their lineage of origin. The window during which ectopic HLH-1 can function is surprisingly broad, spanning the first 3 hours of development when cell lineages are normally established and non-muscle cell fate markers begin to be expressed. We have begun to explore the maternal factors controlling zygotic hlh-1 expression. We find that the Caudal-related homeobox factor PAL-1 can activate hlh-1 in blastomeres that either lack POP-1/TCF or that have down-regulated POP-1/TCF in response to Wnt/MAP kinase signaling. The potent myogenic activity of HLH-1 highlights the remarkable developmental plasticity of early C. elegans blastomeres and reveals the evolutionary conservation of MyoD function.

Transcriptional control and patterning of the pho-1 gene, an essential acid phosphatase expressed in the C. elegans intestine.

We have previously described an acid phosphatase enzyme, PHO-1, present at the lumenal surface of all but the anterior six cells of the Caenorhabditis elegans intestine. In the present paper, we identify the pho-1 structural gene, which encodes a histidine acid phosphatase showing highest similarity to human prostatic acid phosphatase. The pho-1 5'-flanking DNA is capable of directing reporter gene expression that is both gut specific, correctly timed and correctly "patterned", that is, not expressed in the gut anterior. Furthermore, this anterior-posterior patterning of pho-1 expression responds to the C. elegans Wnt pathway as if pho-1 is repressed (directly or indirectly) by high levels of the HMG effector protein POP-1. Transgenic analysis of the pho-1 promoter shows that gut expression is critically dependent on a single WGATAR site. The gut-specific GATA factor ELT-2 binds to this site in vitro and removal of ELT-2 from the embryo destroys expression of the pho-1 reporter. Thus, all our results indicate that pho-1 is a direct downstream target of ELT-2. Finally, the pho-1 loss-of-function mutation shows an interesting and unexpected phenotype for a somatically-expressed hydrolytic enzyme: loss of pho-1 causes arrest of the majority of embryos but this lethality is a maternal effect. We suggest that pho-1 is required by the maternal intestine to assimilate some nutrient or cleavage product that is subsequently provided to the next generation of embryos.

The apical disposition of the Caenorhabditis elegans intestinal terminal web is maintained by LET-413.

We wish to understand how organ-specific structures assemble during embryonic development. In the present paper, we consider what determines the subapical position of the terminal web in the intestinal cells of the nematode Caenorhabditis elegans. The terminal web refers to the organelle-depleted, intermediate filament-rich layer of cytoplasm that underlies the apical microvilli of polarized epithelial cells. It is generally regarded as the anchor for actin rootlets protruding from the microvillar cores. We demonstrate that: (i) the widely used monoclonal antibody MH33 reacts (only) with the gut-specific intermediate filament protein encoded by the ifb-2 gene; (ii) IFB-2 protein accumulates near the gut lumen beginning at the lima bean stage of embryogenesis and remains associated with the gut lumen into adulthood; and (iii) as revealed by immunoelectron microscopy, IFB-2 protein is confined to a discrete circumferential subapical layer within the intestinal terminal web (known in nematodes as the "endotube"); this layer joins directly to the apical junction complexes that connect adjacent gut cells. To investigate what determines the disposition of the IFB-2-containing structure as the terminal web assembles during development, RNAi was used to remove the functions of gene products previously shown to be involved in the overall apicobasal polarity of the developing gut cell. Removal of dlg-1, ajm-1, or hmp-1 function has little effect on the overall position or continuity of the terminal web IFB-2-containing layer. In contrast, removal of the function of the let-413 gene leads to a basolateral expansion of the terminal web, to the point where it can now extend around the entire circumference of the gut cell. The same treatment also leads to concordant basolateral expansion of both gut cell cortical actin and the actin-associated protein ERM-1. LET-413 has previously been shown to be basolaterally located and to prevent the basolateral expansion of several individual apical proteins. In the present context, we conclude that LET-413 is also necessary to maintain the entire terminal web or brush border assembly at the apical surface of C. elegans gut cells, a dramatic example of the so-called "fence" function ascribed to epithelial cell junctions. On the other hand, LET-413 is not necessary to establish this apical location during early development. Finally, the distance at which the terminal web intermediate filament layer lies beneath the gut cell surface (both apical and basolateral) must be determined independently of apical junction position.

Insulinoma-Associated Protein IA-2, a Vesicle Transmembrane Protein, Genetically Interacts with UNC-31/CAPS and Affects Neurosecretion in Caenorhabditis elegans.

IA-2 (insulinoma-associated protein 2), a major autoantigen in type 1 diabetes, is a receptor-tyrosine phosphatase-like protein associated with the membrane of secretory granules of neural and endocrine-specific cells. Loss of IA-2 activity in the mouse results in reduced insulin release and additional phenotypes, consistent with a general effect on neurosecretion and hormone release. To gain further insight into the cellular mechanisms of IA-2 function, we have studied the Caenorhabditis elegans homolog, CeIA-2 encoded by the ida-1 gene. Using two independent putative null alleles of ida-1, we demonstrate that animals lacking CeIA-2 activity are viable and exhibit subtle defects. Genetic studies of mutants in ida-1 and several genes involved in neurosecretory vesicle cargo release and signaling highlight two roles for CeIA-2. First, CeIA-2 has a specific and novel genetic interaction with UNC-31/CAPS, a protein that has been shown in other systems to regulate dense-core vesicle cargo release. Second, loss of CeIA-2 activity enhances weak alleles in the insulin-like signaling pathway. These results suggest that CeIA-2 may be an important factor in dense-core vesicle cargo release with parallels to insulin signaling in mammals.