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The enrichment of intermediate filaments in the apical cytoplasm of intestinal cells is evolutionarily conserved, forming a sheath that is anchored to apical junctions and positioned below the microvillar brush border, which suggests a protective intracellular barrier function. To test this, we used Caenorhabditis elegans , the intestinal cells of which are endowed with a particularly dense intermediate filament-rich layer that is referred to as the endotube. We found alterations in endotube structure and intermediate filament expression upon infection with nematicidal B. thuringiensis or treatment with its major pore-forming toxin crystal protein Cry5B. Endotube impairment due to defined genetic mutations of intermediate filaments and their regulators results in increased Cry5B sensitivity as evidenced by elevated larval arrest, prolonged time of larval development and reduced survival. Phenotype severity reflects the extent of endotube alterations and correlates with reduced rescue upon toxin removal. The results provide in vivo evidence for a major protective role of a properly configured intermediate filament network as an intracellular barrier in intestinal cells. This notion is further supported by increased sensitivity of endotube mutants to oxidative and osmotic stress., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Intermediate filaments are major cytoskeletal components whose assembly into complex networks and isotype-specific functions are still largely unknown. Caenorhabditis elegans provides an excellent model system to study intermediate filament organization and function in vivo. Its intestinal intermediate filaments localize exclusively to the endotube, a circumferential sheet just below the actin-based terminal web. A genetic screen for defects in the organization of intermediate filaments identified a mutation in the catalytic domain of the MAP kinase 7 orthologue sma-5(kc1) In sma-5(kc1) mutants, pockets of lumen penetrate the cytoplasm of the intestinal cells. These membrane hernias increase over time without affecting epithelial integrity and polarity. A more pronounced phenotype was observed in the deletion allele sma-5(n678) and in intestine-specific sma-5(RNAi) Besides reduced body length, an increased time of development, reduced brood size, and reduced life span were observed in the mutants, indicating compromised food uptake. Ultrastructural analyses revealed that the luminal pockets include the subapical cytoskeleton and coincide with local thinning and gaps in the endotube that are often enlarged in other regions. Increased intermediate filament phosphorylation was detected by two-dimensional immunoblotting, suggesting that loss of SMA-5 function leads to reduced intestinal tube stability due to altered intermediate filament network phosphorylation., (© 2016 Geisler et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Background: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide., Results: We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules., Conclusions: The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.

It is commonly accepted that intermediate filaments have an important mechanical function. This function relies not only on intrinsic material properties but is also determined by dynamic interactions with other cytoskeletal filament systems, distinct cell adhesion sites, and cellular organelles which are fine-tuned by multiple signaling pathways. While aspects of these properties and processes can be studied in vitro, their full complexity can only be understood in a viable tissue context. Yet, suitable and easily accessible model systems for monitoring tissue mechanics at high precision are rare. We show that the dissected intestine of the genetic model organism Caenorhabditis elegans fulfills this requirement. The 20 intestinal cells, which are arranged in an invariant fashion, are characterized by a dense subapical mesh of intermediate filaments that are attached to the C. elegans apical junction. We present procedures to visualize details of the characteristic intermediate filament-junctional complex arrangement in living animals. We then report on methods to prepare intestines with a fully intact intermediate filament cytoskeleton and detail procedures to assess their viability. A dual micropipette assay is described to measure mechanical properties of the dissected intestine while monitoring the spatial arrangement of the intermediate filament system. Advantages of this approach are (i) the high reproducibility of measurements because of the uniform architecture of the intestine and (ii) the high degree of accessibility allowing not only mechanical manipulation of an intact tissue but also control of culture medium composition and addition of drugs as well as visualization of cell structures. With this method, examination of worms carrying mutations in the intermediate filament system, its interacting partners and its regulators will become feasible., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The nematode Caenorhabditis elegans is an excellent model system in which to study in vivo organization and function of the intermediate filament (IF) system for epithelial development and function. Using a transgenic ifb-2::cfp reporter strain, a mutagenesis screen was performed to identify mutants with aberrant expression patterns of the IF protein IFB-2, which is expressed in a dense network at the subapical endotube just below the microvillar brush border of intestinal cells. Two of the isolated alleles (kc2 and kc3) were mapped to the same gene, which we refer to as ifo-1 (intestinal filament organizer). The encoded polypeptide colocalizes with IF proteins and F-actin in the intestine. The apical localization of IFO-1 does not rely on IFB-2 but is dependent on LET-413, a basolateral protein involved in apical junction assembly and maintenance of cell polarity. In mutant worms, IFB-2 and IFC-2 are mislocalized in cytoplasmic granules and accumulate in large aggregates at the C. elegans apical junction (CeAJ) in a DLG-1-dependent fashion. Electron microscopy reveals loss of the prominent endotube and disordered but still intact microvilli. Semiquantitative fluorescence microscopy revealed a significant decrease of F-actin, suggesting a general role of IFO-1 in cytoskeletal organization. Furthermore, downregulation of the cytoskeletal organizer ERM-1 and the adherens junction component DLG-1, each of which leads to F-actin reduction on its own, induces a novel synthetic phenotype in ifo-1 mutants resulting in disruption of the lumen. We conclude that IFO-1 is a multipurpose linker between different cytoskeletal components of the C. elegans intestinal terminal web and contributes to proper epithelial tube formation.

The biogenesis of mitochondrial NADH:ubiquinone oxidoreductase (complex I) requires several assembly chaperones. These so-called complex I assembly factors have emerged as a new class of human disease genes. Here, we identified putative assembly factor homologues in Caenorhabditis elegans. We demonstrate that two candidates (C50B8.3/NUAF-1, homologue of NDUFAF1 and R07H5.3/NUAF-3, homologue of NDUFAF3) clearly affect complex I function. Assembly factor deficient worms were shorter, showed a diminished brood size and displayed reduced fat content. Our results suggest that mitochondrial complex I biogenesis is evolutionarily conserved. Moreover, Caenorhabditis elegans appears to be a promising model organism to study assembly factor related human diseases., (Copyright © 2012. Published by Elsevier B.V.)

Caenorhabditis elegans open reading frame T21C9.1 encodes an uncharacterized protein, which is here named MICS-1 (mitochondrial scaffolding protein-1). It is predicted to be the homolog of human outer mitochondrial membrane protein 25 (OMP25 or synaptojanin-2-binding protein), which is a PDZ domain containing protein with a putative role in cellular stress response pathways. Here, we provide evidence that MICS-1 is an interacting partner of mitochondrial protein ATAD-3 (homologue of human ATAD3), which is essential for C. elegans development. We demonstrate that mics-1(RNAi) animals or mics-1 mutants display enhanced longevity with an increased mean lifespan of up to 54% compared to control animals. Of note, also atad-3(RNAi) promoted longevity, although to a lesser extend (29% compared to controls). In addition, thermal stress of mics-1 mutants induced low reactive oxygen species (ROS) production, whereas atad-3(RNAi) animals were highly sensitive to this assay, displaying drastically increased ROS levels. Further studies revealed that MICS-1 and ATAD-3 associated longevity was partially dependent on the presence of DAF-16. However, for both conditions, we also found a DAF-16 independent extension of lifespan. Finally, we observed an additional lifespan extension in mics-1 mutants when subjected to atad-3(RNAi) whereas heat induced ROS production was even aggravated under this condition. This suggests (partially) independent effects of MICS-1 and ATAD-3 on lifespan and ROS production in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The planar cell polarity (PCP) pathway is highly conserved from Drosophila to humans and a PCP-like pathway has recently been described in the nematode Caenorhabditis elegans. The developmental function of this pathway is to coordinate the orientation of cells or structures within the plane of an epithelium or to organize cell-cell intercalation required for correct morphogenesis. Here, we describe a novel role of VANG-1, the only C. elegans ortholog of the conserved PCP component Strabismus/Van Gogh. We show that two alleles of vang-1 and depletion of the protein by RNAi cause an increase of mean life span up to 40%. Consistent with the longevity phenotype vang-1 animals also show enhanced resistance to thermal- and oxidative stress and decreased lipofuscin accumulation. In addition, vang-1 mutants show defects like reduced brood size, decreased ovulation rate and prolonged reproductive span, which are also related to gerontogenes. The germline, but not the intestine or neurons, seems to be the primary site of vang-1 function. Life span extension in vang-1 mutants depends on the insulin/IGF-1-like receptor DAF-2 and DAF-16/FoxO transcription factor. RNAi against the phase II detoxification transcription factor SKN-1/Nrf2 also reduced vang-1 life span that might be explained by gradual inhibition of insulin/IGF-1-like signaling in vang-1. This is the first time that a key player of the PCP pathway is shown to be involved in the insulin/IGF-1-like signaling dependent modulation of life span in C. elegans.

Cell polarity is a fundamental principle guiding development. Early C. elegans embryos contain a diversity of polarized cell types, ranging from asymmetrically dividing stem cells to a polarized epithelium. Over the past two decades, work using C. elegans embryos has led to mechanistic understandings of many aspects of cell polarity establishment, maintenance and propagation. Here we provide basic methods for researchers interested in using C. elegans embryos to study cell polarity, emphasizing the amenability of C. elegans to quantitative molecular analysis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an essential tool for investigating mitochondrial respiratory chain complexes. However, with current BN-PAGE protocols for Caenorhabditis elegans (C. elegans), large worm amounts and high quantities of mitochondrial protein are required to yield clear results. Here, we present an efficient approach to isolate mitochondrial complex I (NADH:ubiquinone oxidoreductase) from C. elegans, grown on agar plates. We demonstrate that considerably lower amounts of mitochondrial protein are sufficient to isolate complex I and to display clear in-gel activity results. Moreover, we present the first complex I assembly profile for C. elegans, obtained by two-dimensional BN/SDS-PAGE., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Understanding how epithelial organs form during morphogenesis is a major problem in developmental biology. In the present paper, we provide a detailed analysis of vang-1, the only homolog of the planar cell polarity protein Strabismus/Van Gogh in Caenorhabditis elegans. We demonstrate that during organogenesis of the intestine, (i) VANG-1 specifically interacts with PDZ 2 domain of DLG-1 (Discs large) and becomes phosphorylated by the kinase domain of the FGF-like receptor tyrosine kinase EGL-15; (ii) VANG-1 is predominantly restrained to the cell cortex but relocates to the apical junction; and (iii) in vang-1 embryos epithelial cells of the intestine are not correctly arranged along the anterior-posterior axis. To investigate what determines the disposition of the VANG-1 protein, either truncated protein forms were expressed in the intestine or RNAi was used to remove the functions of gene products previously shown to be involved in apical junction formation. Removal of the VANG-1 PDZ binding motif "-ESAV" and depletion of dlg-1 or let-413 gene functions interferes with the localization of VANG-1. In addition, egl-15 embryos show a premature relocation of VANG-1 to the apical junction, causing defects that resemble those observed in mutant vang-1 embryos and after intestine-specific overexpression of full-length vang-1. Finally, the localization of VANG-1 depends on DSH-2, a homolog of the planar cell polarity protein Dishevelled and depletion phenocopies vang-1 and egl-15 phenotypes in the embryonic intestine., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Mammalian ATAD3 is a mitochondrial protein, which is thought to play an important role in nucleoid organization. However, its exact function is still unresolved., Results: Here, we characterize the Caenorhabditis elegans (C. elegans) ATAD3 homologue (ATAD-3) and investigate its importance for mitochondrial function and development. We show that ATAD-3 is highly conserved among different species and RNA mediated interference against atad-3 causes severe defects, characterized by early larval arrest, gonadal dysfunction and embryonic lethality. Investigation of mitochondrial physiology revealed a disturbance in organellar structure while biogenesis and function, as indicated by complex I and citrate synthase activities, appeared to be unaltered according to the developmental stage. Nevertheless, we observed very low complex I and citrate synthase activities in L1 larvae populations in comparison to higher larval and adult stages. Our findings indicate that atad-3(RNAi) animals arrest at developmental stages with low mitochondrial activity. In addition, a reduced intestinal fat storage and low lysosomal content after depletion of ATAD-3 suggests a central role of this protein for metabolic activity., Conclusions: In summary, our data clearly indicate that ATAD-3 is essential for C. elegans development in vivo. Moreover, our results suggest that the protein is important for the upregulation of mitochondrial activity during the transition to higher larval stages.

Intermediate filaments (IFs) make up one of the three major fibrous cytoskeletal systems in metazoans. Numerous IF polypeptides are synthesized in cell type-specific combinations suggesting specialized functions. The review concentrates on IFs in the model organism Caenorhabditis elegans which carries great promise to elucidate the still unresolved mechanisms of IF assembly into complex networks and to determine IF function in a living organism. In contrast to Drosophila melanogaster, which lacks cytoplasmic IFs altogether, the nematode genome contains 11 genes coding for cytoplasmic IFs and only a single gene for a nuclear lamin. Its cytoplasmic IFs are expressed in developmentally and spatially defined patterns. As an example we present the case of the intestinal IFs which are abundant in the mechanically resilient endotube, a prominent feature of the C. elegans intestinal terminal web region. This IF-rich structure brings together all three cytoskeletal filaments that are integrated into a coherent entity by the C. elegans apical junction (CeAJ) thereby completely surrounding and stabilizing the intestinal lumen with its characteristic brush border. Concepts on the developmental establishment of the endotube in relation to polarization and its function for maintenance of epithelial integrity are discussed. Furthermore, possible connections of the cytoplasmic cytoskeleton to the nuclear lamin IFs and the importance of these links for nuclear positioning are summarized.

While expectations and applications of nanotechnologies grow exponentially, little is known about interactions of engineered nanoparticles with multicellular organisms. Here we propose the transparent roundworm Caenorhabditis elegans as a simple but anatomically and biologically well defined animal model that allows for whole organism analyses of nanoparticle-bio-interactions. Microscopic techniques showed that fluorescently labelled nanoparticles are efficiently taken up by the worms during feeding, and translocate to primary organs such as epithelial cells of the intestine, as well as secondary organs belonging to the reproductive tract. The life span of nanoparticle-fed Caenorhabditis elegans remained unchanged, whereas a reduction of progeny production was observed in silica-nanoparticle exposed worms versus untreated controls. This reduction was accompanied by a significant increase of the 'bag of worms' phenotype that is characterized by failed egg-laying and usually occurs in aged wild type worms. Experimental exclusion of developmental defects suggests that silica-nanoparticles induce an age-related degeneration of reproductive organs, and thus set a research platform for both, detailed elucidation of molecular mechanisms and high throughput screening of different nanomaterials by analyses of progeny production.

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 let-413/scribble and dlg-1/discs large genes are key regulators of epithelial cell polarity in C. elegans and other systems but the mechanism how they organize a circumferential junctional belt around the apex of epithelial cells is not well understood. We report here that IP(3)/Ca(2+) signaling is involved in the let-413/dlg-1 pathway for the establishment of epithelial cell polarity during the development in C. elegans. Using RNAi to interfere with let-413 and dlg-1 gene functions during post-embryogenesis, we discovered a requirement for LET-413 and DLG-1 in the polarization of the spermathecal cells. The spermatheca forms an accordion-like organ through which eggs must enter to complete the ovulation process. LET-413- and DLG-1-depleted animals exhibit failure of ovulation. Consistent with this phenotype, the assembly of the apical junction into a continuous belt fails and the PAR-3 protein and microfilaments are no longer localized asymmetrically. All these defects can be suppressed by mutations in IPP-5, an inositol polyphosphate 5-phosphatase and in ITR-1, an inositol triphosphate receptor, which both are supposed to increase the intracellular Ca(2+) level. Analysis of embryogenesis revealed that IP(3)/Ca(2+) signaling is also required during junction assembly in embryonic epithelia.

The Caenorhabditis elegans intestinal lumen is surrounded by a dense cytoplasmic network that is laterally attached to the junctional complex and is referred to as the endotube. It localizes to the terminal web region which anchors the microvillar actin filament bundles and is particularly rich in intermediate filaments. To examine their role in intestinal morphogenesis and function, C. elegans reporter strains were generated expressing intestine-specific CFP-tagged intermediate filament polypeptide IFB-2. When these animals were treated with dsRNA against intestinal intermediate filament polypeptide IFC-2, the endotube developed multiple bubble-shaped invaginations that protruded into the enterocytic cytoplasm. The irregularly widened lumen remained surrounded by a continuous IFB-2::CFP-labeled layer. Comparable but somewhat mitigated phenotypic changes were also noted in wild-type N2 worms treated with ifc-2 (RNAi). Junctional complexes were ultrastructurally and functionally normal and the apical domain of intestinal cells was also not altered. These observations demonstrate that IFC-2 is important for structural maintenance of the intestinal tube but is not needed for establishment of the endotube and epithelial cell polarity.

The formation and maintenance of cilia and flagella require a selective and directed transport along the axoneme, a characteristic central bundle of microtubules. Recent evidence suggests an interesting link between the generation of cilia and the protein complexes that establish apico-basal cell polarity.

Members of the ezrin-radixin-moesin (ERM) family of proteins have been found to serve as linkers between membrane proteins and the F-actin cytoskeleton in many organisms. We used RNA interference (RNAi) approach to assay ERM proteins of the Caenorhabditis elegans genome for a possible involvement in apical junction (AJ) assembly or positioning. We identify erm-1 as the only ERM protein required for development and show, by multiple RNA interference, that additional four-point one, ezrin-radixin-moesin (FERM) domain-containing proteins cannot compensate for the depletion of ERM-1. ERM-1 is expressed in most if not all cells of the embryo at low levels but is upregulated in epithelia, like the intestine. ERM-1 protein co-localizes with F-actin and the intermediate filament protein IFB-2 at the apical cell cortex. ERM-1 depletion results in intestine-specific phenotypes like lumenal constrictions or even obstructions. This phenotype arises after epithelial polarization of intestinal cells and can be monitored using markers of the apical junction. We show that the initial steps of epithelial polarization in the intestine are not affected in erm-1(RNAi) embryos but the positioning of apical junction proteins to an apico-lateral position arrests prematurely or fails, resulting in multiple obstructions of the intestinal flow after hatching. Mechanistically, this phenotype might be due to an altered apical cytoskeleton because the apical enrichment of F-actin filaments is lost specifically in the intestine. ERM-1 is the first protein of the apical membrane domain affecting junction remodelling in C. elegans. ERM-1 interacts genetically with the catenin-cadherin system but not with the DLG-1 (Discs large)-dependent establishment of the apical junction.

PGL-1 is a constitutive protein component of C. elegans germ granules, also known as P granules. Maternally supplied PGL-1 is essential for germline development but only at elevated temperature, raising the possibility that redundant factors provide sufficient function at lower temperatures. We have identified two PGL-1-related proteins, PGL-2 and PGL-3, by sequence analysis of the C. elegans genome and by a yeast two-hybrid screen for proteins that interact with PGL-1. PGL-3 is associated with P granules at all stages of development, while PGL-2 is associated with P granules only during postembryonic development. All three PGL proteins interact with each other in vitro. Furthermore, PGL-1 and PGL-3 are co-immunoprecipitated from embryo extracts, indicating that they are indeed in the same protein complex in vivo. Nevertheless, each PGL protein localizes to P granules independently of the other two. pgl-2 or pgl-3 single-mutant worms do not show obvious defects in germline development. However, pgl-1; pgl-3 (but not pgl-2; pgl-1) double-mutant hermaphrodites and males show significantly enhanced sterility at all temperatures, compared to pgl-1 alone. Mutant hermaphrodites show defects in germline proliferation and in production of healthy gametes and viable embryos. Our findings demonstrate that both PGL-2 and PGL-3 are components of P granules, both interact with PGL-1, and at least PGL-3 functions redundantly with PGL-1 to ensure fertility in both sexes of C. elegans.

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.

The Caenorhabditis elegans intestine is a simple and accessible model system to analyze the mechanism of junction assembly. In comparison to Drosophila and vertebrates, the C. elegans apical junction is remarkable because a single electron-dense structure is implicated in complex processes such as epithelial tightness, vectorial transport and cell adhesion. Here we present evidence in support of a heterogeneous molecular assembly of junctional proteins found in Drosophila and vertebrate epithelia associated with different junctions or regions of the plasma membrane. In addition, we show that molecularly diverse complexes participate in different aspects of epithelial maturation in the C. elegans intestine. DLG-1 (Discs large) acts synergistically with the catenin-cadherin complex (HMP-1-HMP-2-HMR-1) and the Ezrin-Radixin-Moesin homolog (ERM-1) to ensure tissue integrity of the intestinal tube. The correct localization of DLG-1 itself depends on AJM-1, a coiled-coil protein. Double depletion of HMP-1 (alpha-catenin) and LET-413 (C. elegans homolog of Drosophila Scribble) suggests that the catenin-cadherin complex is epistatic to LET-413, while additional depletion of subapically expressed CRB-1 (Crumbs) emphasizes a role of CRB-1 concerning apical junction formation in the C. elegans intestine.

The proper segregation of chromosomes during meiosis or mitosis requires the assembly of well organized spindles. In many organisms, meiotic spindles lack centrosomes. The formation of such acentrosomal spindles seems to involve first assembly or capture of microtubules (MTs) in a random pattern around the meiotic chromosomes and then parallel bundling and bipolar organization by the action of MT motors and other proteins. Here, we describe the structure, distribution, and function of KLP-18, a Caenorhabditis elegans Klp2 kinesin. Previous reports of Klp2 kinesins agree that it concentrates in spindles, but do not provide a clear view of its function. During prometaphase, metaphase, and anaphase, KLP-18 concentrates toward the poles in both meiotic and mitotic spindles. Depletion of KLP-18 by RNA-mediated interference prevents parallel bundling/bipolar organization of the MTs that accumulate around female meiotic chromosomes. Hence, meiotic chromosome segregation fails, leading to haploid or aneuploid embryos. Subsequent assembly and function of centrosomal mitotic spindles is normal except when aberrant maternal chromatin is present. This suggests that although KLP-18 is critical for organizing chromosome-derived MTs into a parallel bipolar spindle, the order inherent in centrosome-derived astral MT arrays greatly reduces or eliminates the need for KLP-18 organizing activity in mitotic spindles.

During embryonic development, polarized epithelial cells are either formed during cleavage or formed from mesenchymal cells. Because the formation of epithelia during embryogenesis has to occur with high fidelity to ensure proper development, embryos allow a functional approach to study epithelial cell polarization in vivo. In particular, genetic model organisms have greatly advanced our understanding of the generation and maintenance of epithelial cell polarity. Many novel and important polarity genes have been identified and characterized in invertebrate systems, like Drosophila melanogaster and Caenorhabditis elegans. With the rapid identification of mammalian homologues of these invertebrate polarity genes, it has become clear that many important protein domains, single proteins and even entire protein complexes are evolutionarily conserved. It is to be expected that the field of epithelial cell polarity is just experiencing the 'top of the iceberg' of a large protein network that is fundamental for the specific adhesive, cell signalling and transport functions of epithelial cells.

The polarized nature of epithelial cells is manifested by the nonrandom partitioning of organelles within the cells, the concentration of intercellular junctions at one pole, and the asymmetric distribution of proteins and lipids within the plasma membrane. These features allow epithelia to fulfill their specific tasks, such as targeted uptake and secretion of molecules and the segregation of different tissue compartments. The accessibility of Drosophila melanogaster and Caenorhabditis elegans to genetic and cell biological analyses, combined with the study of mammalian cells in culture, provides an ideal basis for understanding the mechanisms that control the establishment and maintenance of epithelial cell polarity and tissue integrity. Here, we focus on some of the best-studied junctions and membrane-associated protein complexes and their relation to cell polarity. Comparisons between fly, worm, and vertebrate epithelia reveal marked similarities with respect to the molecules used, and pronounced differences in the organization of the junctions themselves.

The correct assembly of junction components, such as E-cadherin and beta-catenin, into the zonula adherens is fundamental for the function of epithelia, both in flies and in vertebrates. In C. elegans, however, the cadherin-catenin system is not essential for general adhesion, raising the question as to the genetic basis controlling junction morphogenesis in nematodes. Here we show that dlg-1, the C. elegans homologue of the Drosophila tumour-suppressor gene discs-large, plays a crucial role in epithelial development. DLG-1 is restricted to adherens junctions of all embryonic epithelia, which contrasts with the localisation of the Drosophila and vertebrate homologues in septate and tight junctions, respectively. Proper localisation of DLG-1 requires the basolateral LET-413 protein, but is independent of the cadherin-catenin system. Embryos in which dlg-1 activity was eliminated by RNA-mediated interference fail to form a continuous belt of junction-associated antigens and arrest development. Loss of dlg-1 activity differentially affects localisation of proteins normally enriched apically to the zonula adherens. While the distribution of an atypical protein kinase C (PKC-3) and other cytoplasmic proteins (PAR-3, PAR-6) is not affected in dlg-1 (RNAi) embryos, the transmembrane protein encoded by crb-1, the C. elegans homologue of Drosophila crumbs, is no longer concentrated in this domain. In contrast to Drosophila, however, crb-1 and a second crb-like gene are not essential for epithelial development in C. elegans. Together the data indicate that several aspects of the spatial organisation of epithelial cells and its genetic control differ between flies, worms, and vertebrates, while others are conserved. The molecular nature of DLG-1 makes it a likely candidate to participate in the organisation of a protein scaffold that controls the assembly of junction components into the zonula adherens., (Copyright 2001 Academic Press.)

Dividing cells need to coordinate the separation of chromosomes with the formation of a cleavage plane. There is evidence that microtubule bundles in the interzone region of the anaphase spindle somehow control both the location and the assembly of the cleavage furrow [1-3]. A microtubule motor that concentrates in the interzone, MKLP1, has previously been implicated in the assembly of both the metaphase spindle and the cleavage furrow [4-6]. To gain insight into mechanisms that might underlie interdependence of the spindle and the cleavage furrow, we used RNA-mediated interference (RNAi) to study the effects of eliminating MKLP1 from Caenorhabditis elegans embryos. Surprisingly, in MKLP1(RNAi) embryos, spindle formation appears normal until late anaphase. Microtubule bundles form in the spindle interzone and the cleavage furrow assembles; anaphase and cleavage furrow ingression initially appear normal. The interzone bundles do not gather into a stable midbody, however, and furrow contraction always fails before complete closure. This sequence of relatively normal mitosis and a late failure of cytokinesis continues for many cell cycles. These and additional results suggest that the interzone microtubule bundles need MKLP1 to encourage the advance and stable closure of the cleavage furrow.

During early nematode embryogenesis a series of asymmetric cleavages in the germ line generates several somatic founder cells and a primordial germ cell. We have found previously that the two soil nematodes Cephalobus spec. and Caenorhabditis elegans express considerable differences in the order of events and spatial arrangement of cells during early embryogenesis. With the help of microinjected fluorescent marker dyes, we show here that these dissimilarities partner major differences in the pattern of intercellular communication. Whilst in C. elegans all early blastomeres become dye-coupled simultaneously, in Cephalobus communication is established progressively in the sequence in which cells are born. In addition, in Cephalobus but not C. elegans, sequential lucifer yellow accumulation indicates stepwise changes in the state of early blastomeres: if injected into the uncleaved zygote, for example, the dye becomes equally distributed to all cells at first but rapidly accumulates in a single blastomere in the 4-cell stage. We speculate that such a redistribution mechanism may be involved in the differential segregation of cytoplasmic components to individual blastomeres. The most dramatic difference between the two species was found with respect to the transfer of high molecular weight molecules. In contrast to C. elegans, in Cephalobus not only small lucifer dyes but also high molecular weight dextrans can diffuse along specific pathways between early somatic cells indicating the presence of large communication channels. However, a transfer of dextran into or out of germ line cells never takes place. The origin of these channels as midbodies of previous mitoses and their potential role for normal development is discussed. Tissue-specific dye-coupling compartments in the slow developing Cephalobus are established in the same order but at a considerably earlier developmental stage than in C. elegans suggesting that this process may depend more on parameters like available time for transcription rather than the number of cell cycles passed through.

During embryogenesis of Caenorhabditis elegans cellular interactions are necessary to determine the fate of blastomeres. In one of these interactions, taking place in the 4-cell stage, the germline cell P2 induces longitudinal orientation of the cleavage spindle in the neighboring EMS cell, its asymmetric division, and the establishment of a gut lineage. Application of several polysulfated hydrocarbon dyes (e.g., trypan blue, TB) in the 1- to 4-cell stages inhibits induction of the gut precursor cell. However, dye application from the late 4-cell stage onward does not interfere with gut induction, supporting the earlier finding of a short time window for this interaction. We also tested the effect of TB on the induction of pharyngeal muscle cells by the MS blastomere, which appears to involve a surface receptor-ligand interaction. We found that this process is inhibited as well. These and additional data indicate that polysulfated hydrocarbon dyes are suitable tools to generally interfere with cell-cell interactions in the nematode embryo.

As more and more cases of necessary cell-cell interactions are revealed, the classical view of mosaic development in nematodes has to be replaced by a more dynamic picture showing different types of intercellular communication. To investigate the pattern and function of communication pathways between cells, we have developed different techniques to shunt fluorescent marker dyes into embryos and hatched animals and study their distribution in vivo. During embryogenesis we find that for a long time all somatic cells form a single dye-coupling compartment while transfer into the germline is restricted already at an early stage. Considerable variations between species with respect to the size of communication channels and the time during which these are functional are observed and can be correlated to differences in the developmental program. A different kind of intracellular communication can be visualized with the help of fluorescent dyes: a transfer of yolk proteins in two phases of the life cycle, in the adult hermaphrodite from the gut into the maturing germ cells, and in the embryo from non-gut cells into the gut primordium. Cell-cell interactions in the nematode embryo can be inhibited with polysulfated hydrocarbon dyes (e.g. Trypan Blue) which bind strongly to the plasma membrane. In summary our data indicate that fluorescent marker dyes can be helpful tools to identify and understand the role of intercellular communication and transfer processes in nematode development.

We have investigated the pattern of cell-cell communication in embryos of the free-living soil nematode Caenorhabditis elegans. For this, we have established a method for microinjection of tracer dyes into individual blastomeres. After iontophoresis of fluorescent dyes of different molecular weights (Lucifer yellow, LY, M(r) 457; rhodamine-labeled dextran, RD, M(r) 4000), we can visualize intercellular communication pathways. The dye-spread of LY, indicating communication via gap junctions, becomes first visible in the late 2-cell stage. From the 4-cell stage onward all cells appear to be well coupled by communication channels, which allow the free diffusion of LY. In contrast, RD remains restricted to the injected cell and its descendants. After the primordial germcell P4 has been generated in the 24-cell stage, dye-spread of LY into this cell and its somatic sister D is delayed. However, the restricted dye-coupling of D is only temporary. After a brief period it joins the somatic compartment. With the beginning of the morphogenesis phase the two existing germline cells (the daughters of P4) are completely uncoupled from the soma, while the latter still forms a single dye-coupling compartment. Only during the second half of embryogenesis different separate somatic communication compartments are established. We followed the pattern of intercellular communication in the alimentary tract and found a progressive restriction into smaller dye-coupling units. Our data are compared to those found in other systems and discussed with respect to cellular determination and differentiation.