mua-6, a gene required for tissue integrity in Caenorhabditis elegans, encodes a cytoplasmic intermediate filament

Locomotion in Caenorhabditis elegans requires force transmission through a network of proteins linking the skeletal muscle, via an intervening basal lamina and epidermis (hypodermis), to the cuticle. Mutations in mua-6 result in hypodermal rupture, muscle detachment from the bodywall, and progressive paralysis. It is shown that mua-6 encodes the cytoplasmic intermediate filament (cIF) A2 protein and that a MUA-6/IFA-2::GFP fusion protein that rescues the presumptive mua-6 null allele localizes to hypodermal hemidesmosomes. This result is consistent with what is known about the function of cIFs in vertebrates. Although MUA-6/IFA-2 is expressed embryonically, and plays an essential postembryonic role in tissue integrity, it is not required for embryonic development of muscle-cuticle linkages nor for the localization of other cIFs or hemidesmosome-associated proteins in the embryo. Finally, the molecular lesion in the mua-6(rh85) allele suggests that the head domain of the MUA-6/IFA-2 is dispensable for its function.     

Functional analysis of the single calmodulin gene in the nematode Caenorhabditis elegans by RNA interference and 4-D microscopy

Calmodulin (CaM), a small calcium-binding protein, is the key mediator of numerous calcium-induced changes in cellular activity. Its ligands include enzymes, cytoskeletal proteins and ion channels, identified in large part by biochemical and cell biological approaches. Thus far it has been difficult to assess the function of CaM genetically, because of the maternal supply in Drosophila and the presence of at least three nonallelic genes in vertebrates. Here we use the unique possibility offered by the C. elegans model system to inactivate the single CaM gene (cmd-1) through RNA interference (RNAi). We show that the RNAi microinjection approach results in a severe embryonic lethal phenotype. Embryos show disturbed morphogenesis, aberrant cell migration patterns, a striking hyperproliferation of cells and multiple defects in apoptosis. Finally, we show that RNAi delivery by the feeding protocol does not allow the efficient silencing of the CaM gene obtained by microinjection. General differences between the two delivery methods are discussed.     

In vivo and in vitro evidence that the four essential intermediate filament (IF) proteins A1, A2, A3 and B1 of the nematode Caenorhabditis elegans form an obligate heteropolymeric IF system

The in vitro polymerization and tissue-specific expression patterns of the four essential intermediate filament (IF) proteins (A1, A2, A3, B1) and the non-essential IF protein A4 were analyzed. Recombinant B1, used as a probe in blot overlay assays of the 11 Caenorhabditis elegans IF proteins, reacted strongly with proteins A1 to A4, indicating a heterotypic interaction. Obligate heteropolymeric filament assembly in vitro was confirmed by electron microscopy. Protein B1 formed long IF when mixed with an equimolar amount of A1, A2 or A3. Developmentally regulated coexpression of B1 and one or more members of the A family was found with GFP-promoter reporters. This coexpression pattern argues for a heteropolymer system in vivo. One or both splice variants of the B1 gene are always coexpressed in a tissue-specific manner with at least one member of the A family in hypodermis, pharynx, pharyngeal-intestinal valve, excretory cells, uterus, vulva and rectum. Interestingly, while the intestine normally lacks a B1/A pair, the dauer larva shows intestinal B1 and A4. These results are in line with similar postembryonic phenotypes of the hypodermis induced by RNA interference (RNAi) of genes B1, A2 and A3. Similarly, defects of the pharynx and its A1-GFP containing tonofilaments observed in the postembryonic B1 RNAi phenotype are consistent with the coexpression of B1 and A1 in the marginal cells. Thus RNAi analyses provide independent evidence for the existence of the B1/A obligate heteropolymer system in vivo. Proteins A1 and B1 have a similar and rather slow turnover rate in photobleaching experiments of the pharynx tonofilaments.     

The cephalochordate Branchiostoma genome contains 26 intermediate filament (IF) genes: Implications for evolution of chordate IF proteins

We analyzed the draft genome of the cephalochordate Branchiostoma floridae (B. floridae) for genes encoding intermediate filament (IF) proteins. From 26 identified IF genes 13 were not reported before. Four of the new IF genes belong to the previously established Branchiostoma IF group A, four to the Branchiostoma IF group B, one is homologous to the type II keratin E2 while the remaining four new IF sequences N1 to N4 could not be readily classified in any of the previously established Branchiostoma IF groups. All eleven identified A and B2-type IF genes are located on the same genomic scaffold and arose due to multiple cephalochordate-specific duplications. Another IF gene cluster, identified in the B. floridae genome, contains three keratins (E1, Y1, D1), two keratin-like IF genes (C2, X1), one new IF gene (N1) and one IF unrelated gene, but does not show any similarities to the well defined vertebrate type I or type II keratin gene clusters. In addition, some type III sequence features were documented in the new IF protein N2, which, however, seems to share a common ancestry with the Branchiostoma keratins D1 and two keratin-related genes C. Thus, a few type I and type II keratin genes existed in a common ancestor of cephalochordates and vertebrates, which after separation of these two lineages gave rise to the known complexities of the vertebrate cytoplasmic type I–IV IF proteins, as well as to the multiple keratin and related IF genes in cephalochordates, due to multiple gene duplications, deletions and sequence divergences.     

Analysis of RING finger genes required for embryogenesis in C. elegans

The RING finger motif exists in E3 ligases of the ubiquitination pathway. These ubiquitin ligases bind to target proteins, leading to their modification by covalent addition of ubiquitin peptides. Current databases contain hundreds of proteins with RING finger motifs. This study investigates the role of RING finger genes in embryogenesis of the nematode, Caenorhabditis elegans. We expand the previous list of RING finger-containing genes and show that there are 103 RING finger-containing genes in the C. elegans genome. DNA microarrays of these 103 genes were probed with various RNA samples to identify 16 RING finger genes whose expression is enriched in the germline. RNA interference (RNAi) analysis was then used to determine the developmental role of these genes. One RING finger gene, C32D5.10, showed a dramatic larval arrest upon RNAi. Three RING finger genes exhibited embryonic lethality after RNAi. These three genes include par-2, and two small RING finger proteins: F35G12.9 (an ortholog of APC11) and ZK287.5 (an ortholog of rbx1). Embryos from RNAi of the APC11 and rbx1 orthologs were arrested in the cell cycle, confirming the role of these particular RING finger proteins in regulation of the cell cycle. genesis 38:1-12, 2004.     

Expression profiles of the essential intermediate filament (IF) protein A2 and the IF protein C2 in the nematode Caenorhabditis elegans

The multigene family of intermediate filament (IF) proteins in Caenorhabditis elegans covers 11 members of which four (A1-3, B1) are essential for development. Suppression of a fifth gene (C2) results in a dumpy phenotype. Expression patterns of three essential genes (A1, A3, B1) were already reported. To begin to analyze the two remaining RNAi phenotypes we followed the expression of the A2 and C2 proteins. Expression of A2 mRNA starts in larval stage L1 and continues in the adult. Transgenic A2 promoter/gfp larvae strongly display GFP in the main body hypodermis but not in seam cells. This pattern and the muscle displacement/paralysis induced by RNAi silencing are consistent with the role of this protein in keeping the correct hypodermis/muscle relationship during development. IF protein C2 occurs in the cytoplasm and desmosomes of intestinal cells and in pharynx desmosomes. Expression of C2 starts in the late embryo and persists in all further stages.     

Inhibitory effects of caffeine on gustatory plasticity in the nematode Caenorhabditis elegans

The effects of caffeine on salt chemotaxis learning were investigated using the nematode Caenorhabditis elegans. To estimate the degree of salt chemotaxis learning, nematodes were placed in a mixed solution of NaCl and caffeine, and then the chemotaxis index of NaCl was obtained from the nematodes placed on agar medium after pre-exposure to caffeine concentrations of 0.01, 0.1, 0.3, and 1.0%. Locomotor activity and preference behavior for caffeine were also estimated under these caffeine conditions. Nematodes pre-exposed to 0.3% caffeine showed inhibition of salt chemotaxis learning. Additional experiments indicated that nematodes showed a preference response to the middle concentration of caffeine (0.1%), with preference behavior declining in the 0.3% caffeine condition. Stable locomotor activity was observed under 0.01–0.3% caffeine conditions. These results suggest that salt chemotaxis learning with 0.3% caffeine is useful for investigating the effects of caffeine on learning in nematodes.     

The influence of metabolic rate on longevity in the nematode Caenorhabditis elegans

Much of the recent interest in aging research is due to the discovery of genes in a variety of model organisms that appear to modulate aging. A large amount of research has focused on the use of such long-lived mutants to examine the fundamental causes of aging. While model organisms do offer many advantages for studying aging, it also critical to consider the limitations of these systems. In particular, ectothermic (poikilothermic) organisms can tolerate a much larger metabolic depression than humans. Thus, considering only chronological longevity when assaying for long-lived mutants provides a limited perspective on the mechanisms by which longevity is increased. In order to provide true insight into the aging process additional physiological processes, such as metabolic rate, must also be assayed. This is especially true in the nematode Caenorhabditis elegans, which can naturally enter into a metabolically reduced state in which it survives many times longer than its usual lifetime. Currently it is seen as controversial if long-lived C. elegans mutants retain normal metabolic function. Resolving this issue requires accurately measuring the metabolic rate of C. elegans under conditions that minimize environmental stress. Additionally, the relatively small size of C. elegans requires the use of sensitive methodologies when determining metabolic rates. Several studies indicating that long-lived C. elegans mutants have normal metabolic rates may be flawed due to the use of inappropriate measurement conditions and techniques. Comparisons of metabolic rate between long-lived and wild-type C. elegans under more optimized conditions indicate that the extended longevity of at least some long-lived C. elegans mutants may be due to a reduction in metabolic rate, rather than an alteration of a metabolically independent genetic mechanism specific to aging.     

The sequence of a cytoplasmic intermediate filament (IF) protein from theannelid Lumbricus terrestris emphasizes a distinctive feature of protostomic IF proteins

The complete cDNA clone for a cytoplasmic intermidiatefilament (IF) protein from the annelid Lumbricus terrestris reported here, shows an extra 42 residues in the coil 1b subdomain of the central rod, as do the IF proteins from nematodes and moluscs. These extra six heptads are also present in all nuclear lamins but not in any known vertebrate cytoplasmic IF protein. Thus, it seems that protostomic metazoa conserve a lamin-like structural element in their cytoplasmic IF proteins, which was lost in the deuterostomic metazoan branch leading to the vertebrates.     

Structure of an invertebrate gene encoding cytoplasmic intermediate filament (IF) proteins: implications for the origin and the diversification of IF proteins.

The structure of the single gene encoding the cytoplasmic intermediate filament (IF) proteins in non-neuronal cells of the gastropod Helix aspersa is described. Genomic and cDNA sequences show that the gene is composed of 10 introns and 11 exons, spanning greater than 60 kb of DNA. Alternative RNA processing accounts for two mRNA families which encode two IF proteins differing only in their C-terminal sequence. The intron/exon organization of the Helix rod domain is identical to that of the vertebrate type III IF genes in spite of low overall protein sequence homology and the presence of an additional 42 residues in coil 1b of the invertebrate sequence. Intron position homology extends to the entire coding sequence comprising both the rod and tail domains when the invertebrate IF gene is compared with the nuclear lamin LIII gene of Xenopus laevis presented in the accompanying report of Döring and Stick. In contrast the intron patterns of the tail domains of the invertebrate IF and the lamin genes differ from those of the vertebrate type III genes. The combined data are in line with an evolutionary descent of cytoplasmic IF proteins from a nuclear lamin-like progenitor and suggest a mechanism for this derivation. The unique position of intron 7 in the Helix IF gene indicates that the archetype IF gene arose by the elimination of the nuclear localization sequence due to the recruitment of a novel splice site. The presumptive structural organization of the archetype IF gene allows predictions with respect to the later diversification of metazoan IF genes. Whereas models proposing a direct derivation of neurofilament genes seem unlikely, the earlier speculation of an mRNA transposition mechanism is compatible with current results.     

Two types of chloride transporters are required for GABA(A) receptor-mediated inhibition in C. elegans

Chloride influx through GABA-gated Cl(-) channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl(-) gradient established in part by K(+)-Cl(-) cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na(+)-driven Cl(-)-HCO(3)(-) exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K(+)-Cl(-) cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl(-) extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl(-) gradient such that Cl(-) flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl(-) gradient. Our results show that Na(+)-driven Cl(-)-HCO(3)(-) exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.     

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.     

Molecular and functional analysis of apical junction formation in the gut epithelium of Caenorhabditis elegans

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 identification and localization of two intermediate filament proteins in the tunic of Styela plicata (Tunicata, Styelidae)

The intermediate filament (IF) proteins Styela C and Styela D from the tunicate Styela (Urochordata) are co-expressed in all epidermal cells and they are thought to behave as type I and type II keratins. These two IF proteins, Styela C and Styela D, were identified in immunoblots of proteins isolated from the tunic of Styela plicata. The occurrence and distribution of these proteins within the tunic of this ascidian was examined by means of immunofluorescence and immunoperoxidase techniques, using anti-Styela C and anti-Styela D antibodies. In addition, immuno-electron microscopy of the tunic showed that the two proteins are located in the cuticle layer and in the tunic matrix. These results represent the first data about the presence of IF proteins in the tunic of adult ascidian S. plicata. The possible involvement of these IF proteins in reinforcing the integrity of the tunic, that represents the interface between the animal body and the external environment, is discussed.     

Cytochrome P450-dependent metabolism of eicosapentaenoic acid in the nematode Caenorhabditis elegans

The genome of Caenorhabditis elegans contains 75 full length cytochrome P450 (CYP) genes whose individual functions are largely unknown yet. We tested the hypothesis that some of them may be involved in the metabolism of eicosapentaenoic acid (EPA), the predominant polyunsaturated fatty acid of this nematode. Microsomes isolated from adult worms contained spectrally active CYP proteins and showed NADPH-CYP reductase (CPR) activities. They metabolized EPA and with lower activity also arachidonic acid (AA) to specific sets of regioisomeric epoxy- and ω-/(ω-1)-hydroxy-derivatives. 17(R),18(S)-epoxyeicosatetraenoic acid was produced as the main EPA metabolite with an enantiomeric purity of 72%. The epoxygenase and hydroxylase reactions were NADPH-dependent, required the functional expression of the CPR-encoding emb-8 gene, and were inhibited by 17-ODYA and PPOH, two compounds known to inactivate mammalian AA-metabolizing CYP isoforms. Multiple followed by single RNAi gene silencing experiments identified CYP-29A3 and CYP-33E2 as the major isoforms contributing to EPA metabolism in C. elegans. Liquid chromatography/mass spectrometry revealed that regioisomeric epoxy- and hydroxy-derivatives of EPA and AA are endogenous constituents of C. elegans. The endogenous EPA metabolite levels were increased by treating the worms with fenofibrate, which also induced the microsomal epoxygenase and hydroxylase activities. These results demonstrate for the first time that C. elegans shares with mammals the capacity to produce CYP-dependent eicosanoids and may thus facilitate future studies on the mechanisms of action of this important class of signaling molecules.     

More mog genes that influence the switch from spermatogenesis to oogenesis in the hermaphrodite germ line of Caenorhabditis elegans

The Caenorhobditis elegans XX animal possesses a hermaphrodite germ line, producing first sperm, then oocytes. In this paper, we report the genetic identification of five genes, mog-2, mog-3, mog-4, mog-5, and mog-6, that influence the hermaphrodite switch from sper-matogenesis to oogenesis. In mcg-2-mog-6 mutants, spermatogenesis continues past the time at which hermaphrodites normally switch into oogenesis and no oocytes are observed. Therefore, in these mutants, germ cells are transformed from a female fate (oocyte) to a male fate (sperm). The fem-3 gene is one of five genes that acts at the end of the germline sex determination pathway to direct spermatogenesis. Analyses of mog;fem-3 double mutants suggest that the mog-2-mog-6 genes act before fem-3; thus these genes may be in a position to negatively regulate fem-3 or one of the other terminal regulators of germline sex determination. Double mutants of fem-3 and any one of the mog mutations make oocytes. Using these double mutants, we show that oocytes from any mog;fem-3 double mutant are defective in their ability to support embryogenesis. This maternal effect lethality indicates that each of the mog genes is required for embryogenesis. The two defects in mog-2-mog-6 mutants are similar to those of mog-1: all six mog genes eliminate the sperm/oocyte switch in hermaphrodites and cause maternal effect lethality. We propose that the mog-2-mog-6 mutations identify genes that act with mog-1 to effect the sperm/oocyte switch. We further speculate that the mog-1-mog-6 mutations all interfere with translational controls of fem-3 and other maternal mRNAs. © 1993 Wiley-Liss, Inc.     

Metabolism, physiology and stress defense in three aging Ins/IGF-1 mutants of the nematode Caenorhabditis elegans

The insulin/insulin-like growth factor-1 (Ins/IGF-1) pathway regulates the aging rate of the nematode Caenorhabditis elegans. We describe other features of the three Ins/IGF-1 mutants daf-2, age-1 and aap-1. We show that the investigated Ins/IGF-1 mutants all have a reduced body volume, reduced reproductive capacity, increased ATP concentrations and an elevated stress resistance. We also observed that heat production is lower in these mutants, although the respiration rate was similar or higher compared with wild-type individuals, suggesting a metabolic shift in these mutants.     

Calcium-sensitive activity and conformation of Caenorhabditis elegans gelsolin-like protein 1 are altered by mutations in the first gelsolin-like domain

The gelsolin family of actin regulatory proteins is activated by Ca(2+) to sever and cap actin filaments. Gelsolin has six homologous gelsolin-like domains (G1-G6), and Ca(2+)-dependent conformational changes regulate its accessibility to actin. Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) has only four gelsolin-like domains (G1-G4) and still exhibits Ca(2+)-dependent actin filament-severing and -capping activities. We found that acidic residues (Asp-83 and Asp-84) in G1 of GSNL-1 are important for its Ca(2+) activation. These residues are conserved in GSNL-1 and gelsolin and previously implicated in actin-severing activity of the gelsolin family. We found that alanine mutations at Asp-83 and Asp-84 (D83A/D84A mutation) did not disrupt actin-severing or -capping activity. Instead, the mutants exhibited altered Ca(2+) sensitivity when compared with wild-type GSNL-1. The D83A/D84A mutation enhanced Ca(2+) sensitivity for actin severing and capping and its susceptibility to proteolytic digestion, suggesting a conformational change. Single mutations caused minimal changes in its activity, whereas Asp-83 and Asp-84 were required to stabilize Ca(2+)-free and Ca(2+)-bound conformations, respectively. On the other hand, the D83A/D84A mutation suppressed sensitivity of GSNL-1 to phosphatidylinositol 4,5-bisphosphate inhibition. The structure of an inactive form of gelsolin shows that the equivalent acidic residues are in close contact with G3, which may maintain an inactive conformation of the gelsolin family.