Whole-genome RNAi screen identifies methylation-related genes influencing lipid metabolism in Caenorhabditis elegans

Lipid droplets (LDs) are highly conserved multifunctional cellular organelles and aberrant lipid storage in LDs can lead to many metabolic diseases. However, the molecular mechanisms governing lipid dynamic changes remain elusive, and the high-throughput screen of genes influencing LD morphology was limited by lacking specific LD marker proteins in the powerful genetic tool Caenorhabditis elegans. In this study, we established a new method to conduct whole-genome RNAi screen using LD resident protein DHS-3 as a LD marker, and identified 78 genes involved in significant LD morphologic changes. Among them, mthf-1, as well as a series of methylation-related genes, was found dramatically influencing lipid metabolism. SREBP-1 and SCD1 homologs in C. elegans were involved in the lipid metabolic change of mthf-1(RNAi) worms, and the regulation of ATGL-1 also contributed to it by decreasing triacylglycerol (TAG) hydrolysis. Overall, this study not only identified important genes involved in LD dynamics, but also provided a new tool for LD study using C. elegans, with implications for the study of lipid metabolic diseases.     

Yolk proteins of Caenorhabditis elegans

A group of proteins judged on several criteria to be yolk proteins have been isolated from a homogenate of the nematode Caenorhabditis elegans. Comparison of partial proteolysis fragments indicates that the two bands of a 170,000-dalton doublet (yp170) are closely related; bands observed at 115,000 daltons (yp115) and 88,000 daltons (yp88) appear to be structurally distinct. All three yolk protein species are glycoproteins, as judged by binding of the lectin concanavalin A. The yp170 doublet has been purified by gel filtration in the presence of sodium dodecyl sulfate. An antiserum obtained by immunization with the purified yp170 doublet does not bind either of the two smaller proteins. Staining of C. elegans eggs by indirect immunofluorescence with the anti-yp170 serum indicates a dispersed cytoplasmic location for the antigen throughout embryogenesis, with apparent segregation to the intestine immediately prior to hatching.     

New roles for acyl-CoA-binding proteins (ACBPs) in plant development, stress responses and lipid metabolism

ACBPs are implicated in acyl-CoA trafficking in many eukaryotes and some prokaryotes. Six genes encode proteins designated as AtACBP1-AtACBP6 in the Arabidopsis thaliana ACBP family. These ACBPs are conserved in the acyl-CoA-binding domain, but vary in size from 92 amino acids (10.4 kDa) to 668 amino acids (73.1 kDa), and are subcellularly localised to different compartments in plant cells. Results from in vitro binding assays show that their corresponding recombinant proteins exhibit differential binding affinities to acyl-CoA esters and phospholipids, implying that these ACBPs may have non-redundant biological functions in vivo. By using knockout/downregulated and overexpression lines of Arabidopsis ACBPs, recent investigations have revealed that in addition to their proposed roles in phospholipid metabolism, these ACBPs can influence plant development including early embryogenesis and leaf senescence, as well as plant stress responses including heavy metal resistance, oxidative stress, freezing tolerance and pathogen resistance. In this review, recent progress on the biochemical and functional analyses of Arabidopsis ACBPs, their links to metabolic/signalling pathways, and their potential applications in development of stress tolerance are discussed.     

MAA-1, a novel acyl-CoA-binding protein involved in endosomal vesicle transport in Caenorhabditis elegans

The budding and fission of vesicles during membrane trafficking requires many proteins, including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted by the hydrolysis of acyl-CoA lipid esters. The mechanisms by which these lipid esters are directed to the appropriate membranes in vivo, and their precise roles in vesicle biogenesis, are not yet understood. Here, we present the first report on membrane associated ACBP domain-containing protein-1 (MAA-1), a novel membrane-associated member of the acyl-CoA-binding protein family. We show that in Caenorhabditis elegans, MAA-1 localizes to intracellular membrane organelles in the secretory and endocytic pathway and that mutations in maa-1 reduce the rate of endosomal recycling. A lack of maa-1 activity causes a change in endosomal morphology. Although in wild type, many endosomal organelles have long tubular protrusions, loss of MAA-1 activity results in loss of the tubular domains, suggesting the maa-1 is required for the generation or maintenance of these domains. Furthermore, we demonstrate that MAA-1 binds fatty acyl-CoA in vitro and that this ligand-binding ability is important for its function in vivo. Our results are consistent with a role for MAA-1 in an acyl-CoA-dependent process during vesicle formation.     

Nuclear lipid droplets and nuclear damage in Caenorhabditis elegans

Fat stored in the form of lipid droplets has long been considered a defining characteristic of cytoplasm. However, recent studies have shown that nuclear lipid droplets occur in multiple cells and tissues, including in human patients with fatty liver disease. The function(s) of stored fat in the nucleus has not been determined, and it is possible that nuclear fat is beneficial in some situations. Conversely, nuclear lipid droplets might instead be deleterious by disrupting nuclear organization or triggering aggregation of hydrophobic proteins. We show here that nuclear lipid droplets occur normally in C. elegans intestinal cells and germ cells, but appear to be associated with damage only in the intestine. Lipid droplets in intestinal nuclei can be associated with novel bundles of microfilaments (nuclear actin) and membrane tubules that might have roles in damage repair. To increase the normal, low frequency of nuclear lipid droplets in wild-type animals, we used a forward genetic screen to isolate mutants with abnormally large or abundant nuclear lipid droplets. Genetic analysis and cloning of three such mutants showed that the genes encode the lipid regulator SEIP-1/seipin, the inner nuclear membrane protein NEMP-1/Nemp1/TMEM194A, and a component of COPI vesicles called COPA-1/α-COP. We present several lines of evidence that the nuclear lipid droplet phenotype of copa-1 mutants results from a defect in retrieving mislocalized membrane proteins that normally reside in the endoplasmic reticulum. The seip-1 mutant causes most germ cells to have nuclear lipid droplets, the largest of which occupy more than a third of the nuclear volume. Nevertheless, the nuclear lipid droplets do not trigger apoptosis, and the germ cells differentiate into gametes that produce viable, healthy progeny. Thus, our results suggest that nuclear lipid droplets are detrimental to intestinal nuclei, but have no obvious deleterious effect on germ nuclei.     

Developmental regulation of energy metabolism in Caenorhabditis elegans

Changes in energy metabolism during larval development in Caenorhabditis elegans have been investigated using phosphorus nuclear magnetic resonance (31P NMR). The relative concentrations of ATP, ADP, AMP, sugar phosphates, and other metabolites were observed to change during larval development, producing stage-specific spectra. These spectra are consistent with enzyme assays for isocitrate dehydrogenase and isocitrate lyase, indicating that high activity of the glyoxylate pathway during embryonic development decreases during the first larval (L1) stage, and respiration during the L2, L3, and L4 stages occurs preferentially through the TCA cycle. Metabolic strategies were further studied using mutants that are predisposed to enter the dauer stage, a developmentally arrested third-stage larva formed under conditions of overcrowding and limited food. After the L1 molt, energy metabolism in animals destined to become dauer larvae diverges from that of animals committed to growth. Relative to the L1, the L2 larvae committed to growth exhibit increased isocitrate dehydrogenase activity as well as increases in ATP and other high-energy phosphates, but predauer (L2d) larvae exhibit declining enzyme activities and declining levels of high-energy phosphates. The predominant phosphorus NMR signal in dauer larva extracts corresponds to inorganic phosphate. We conclude that metabolism is regulated during C. elegans larval development, with a major transition apparent after the L1 stage. This transition does not occur in larvae destined to form dauer larvae.     

Mechanisms of aging and energy metabolism in Caenorhabditis elegans

Aging studies on diverse species ranging from yeast to man have culminated in the delineation of several signaling pathways that influence the process of senescent decline and aging. While understanding these interlinked signal-transduction cascades is becoming even more detailed and comprehensive, the cellular and biochemical processes they impinge upon to modulate the rate of senescent decline and aging have lagged considerably behind. This fundamental question is one of the most important challenges of modern aging research and has been the focus of recent research efforts. Emerging findings provide insight into the facets of cellular metabolism which can be fine-tuned by upstream signaling events to ultimately promote longevity. Here, we survey the mechanisms regulating aging in the simple nematode worm Caenorhabditis elegans, aiming to highlight recent discoveries that shed light into the interface between aging signaling pathways and cellular energy metabolism. Our objective is to review the current understanding of the processes involved and discuss mechanisms that are likely conserved in higher organisms.     

Uncovering new functions for microRNAs in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, microRNA (miRNA) regulation of development was first observed in the striking abnormalities of lin-4 and let-7 loss of function mutants. However, after these first two miRNA mutant phenotypes were described, progress on the identification of miRNA functions in worms slowed considerably. Recent advances reveal new functions for miRNAs in embryonic and larval development as well as in the regulation of lifespan and stress response. Results from a combination of?computational, biochemical, and genetic approaches have deepened our understanding of miRNA regulation of target mRNAs and support the hypothesis that miRNAs have an important role in ensuring the robustness of developmental and physiological pathways.     

The mutational structure of metabolism in Caenorhabditis elegans

A properly functioning organism must maintain metabolic homeostasis. Deleterious mutations degrade organismal function, presumably at least in part via effects on metabolic function. Here we present an initial investigation into the mutational structure of the Caenorhabditis elegans metabolome by means of a mutation accumulation experiment. We find that pool sizes of 29 metabolites vary greatly in their vulnerability to mutation, both in terms of the rate of accumulation of genetic variance (the mutational variance, VM) and the rate of change of the trait mean (the mutational bias, ΔM). Strikingly, some metabolites are much more vulnerable to mutation than any other trait previously studied in the same way. Although we cannot statistically assess the strength of mutational correlations between individual metabolites, principal component analysis provides strong evidence that some metabolite pools are genetically correlated, but also that there is substantial scope for independent evolution of different groups of metabolites. Averaged over mutation accumulation lines, PC3 is positively correlated with relative fitness, but a model in which metabolites are uncorrelated with fitness is nearly as good by Akaike's Information Criterion.     

Tissue-specific synthesis of yolk proteins in Caenorhabditis elegans

The primary site of yolk protein synthesis in the nematode, Caenorhabditis elegans, has been determined. In animals containing no gonadal cells (obtained by laser ablation of the gonadal precursor cells early in development), yolk proteins are present in abundance. This demonstrates that yolk proteins are made outside the gonad. An examination of proteins present in tissues isolated by dissection, and a comparison of proteins synthesized by isolated tissues incubated in vitro have identified the intestine as the major site of yolk protein synthesis. We propose that yolk proteins are synthesized in the intestine, secreted from the intestine into the body cavity, and taken up from the body cavity by the gonad to reach oocytes. The site of yolk protein synthesis has also been examined in four mutants that have largely male somatic tissues, but a hermaphrodite germ line. Here again, yolk proteins are produced by intestines in a hermaphrodite-specific manner. This suggests that sex determination is coordinately regulated in intestinal and germ line tissues.     

Trehalose metabolism genes in Caenorhabditis elegans and filarial nematodes

The sugar trehalose is claimed to be important in the physiology of nematodes where it may function in sugar transport, energy storage and protection against environmental stresses. In this study we investigated the role of trehalose metabolism in nematodes, using Caenorhabditis elegans as a model, and also identified complementary DNA clones putatively encoding genes involved in trehalose pathways in filarial nematodes. In C. elegans two putative trehalose-6-phosphate synthase (tps) genes encode the enzymes that catalyse trehalose synthesis and five putative trehalase (tre) genes encode enzymes catalysing hydrolysis of the sugar. We showed by RT-PCR or Northern analysis that each of these genes is expressed as mRNA at all stages of the C. elegans life cycle. Database searches and sequencing of expressed sequence tag clones revealed that at least one tps gene and two tre genes are expressed in the filarial nematode Brugia malayi, while one tps gene and at least one tre gene were identified for Onchocerca volvulus. We used the feeding method of RNA interference in C. elegans to knock down temporarily the expression of each of the tps and tre genes. Semiquantitative RT-PCR analysis confirmed that expression of each gene was silenced by RNA interference. We did not observe an obvious phenotype for any of the genes silenced individually but gas-chromatographic analysis showed >90% decline in trehalose levels when both tps genes were targeted simultaneously. This decline in trehalose content did not affect viability or development of the nematodes.     

Cadherin superfamily proteins in Caenorhabditis elegans and Drosophila melanogaster

The ability to form selective cell-cell adhesions is an essential property of metazoan cells. Members of the cadherin superfamily are important regulators of this process in both vertebrates and invertebrates. With the advent of genome sequencing projects, determination of the full repertoire of cadherins available to an organism is possible and here we present the identification and analysis of the cadherin repertoires in the genomes of Caenorhabditis elegans and Drosophila melanogaster. Hidden Markov models of cadherin domains were matched to the protein sequences obtained from the translation of the predicted gene sequences. Matches were made to 21 C. elegans and 18 D. melanogaster sequences. Experimental and theoretical work on C. elegans sequences, and data from ESTs, show that three pairs of genes, and two triplets, should be merged to form five single genes. It also produced sequence changes at one or both of the 5' and 3' termini of half the sequences. In D. melanogaster it is probable that two of the cadherin genes should also be merged together and that three cadherin genes should be merged with other neighbouring genes.Of the 15 cadherin proteins found in C. elegans, 13 have the features of cell surface proteins, signal sequences and transmembrane helices; the other two have only signal sequences. Of the 17 in D. melanogaster, 11 at present have both features and another five have transmembrane helices. The evidence currently available suggests about one-third of the cadherins in the two organisms can be grouped into subfamilies in which all, or parts of, the molecules are conserved. Each organism also has a approximately 980 residue protein (CDH-11 and CG11059) with two cadherin domains and whose sequences match well over their entire length two proteins from human brain. Two proteins in C. elegans, HMR-1A and HMR-1B, and three in D. melanogaster, CadN, Shg and CG7527, have cytoplasmic domains homologous to those of the classical cadherin genes of chordates but their extracellular regions have different domain structures. Other common subclasses include the seven-helix membrane cadherins, Fat-like protocadherins and the Ret-like cadherins. At present, the remaining cadherins have no obvious similarities in their extracellular domain architecture or homologies to their cytoplasmic domains and may, therefore, represent species-specific or phylum-specific molecules.     

The Caenorhabditis elegans sqv genes and functions of proteoglycans in development

In the nematode Caenorhabditis elegans, the vulva is a simple tubular structure linking the gonads with the external cuticle. In this review we summarize knowledge of inter- and intracellular signaling during vulval development and of the genes required for vulval invagination. Mutants of one set of these genes, the sqv genes, have a normal number of vulval precursor cells (VPCs) with an unperturbed cell lineage but the invagination space, normally a tube, is either collapsed or absent. We review evidence that the sqv genes are involved in glycosaminoglycan synthesis and speculate on ways in which defective glycosaminoglycan formation might lead to collapse of the vulval structure.     

Defective arginine metabolism impairs mitochondrial homeostasis in Caenorhabditis elegans

Arginine catabolism involves enzyme-dependent reactions in both mitochondria and the cytosol,defects in which may lead to hyperargininemia,a devastating developmental disorder.It is largely unknown if defective arginine catabolism has any effects on mitochondria.Here we report that normal arginine catabolism is essential for mitochondrial homeostasis in Caenorhabditis elegans.Mutations of the arginase gene argn-1 lead to abnormal mitochondrial enlargement and reduced adenosine triphosphate(ATP) production in C elegans hypodermal cells.ARGN-1 localizes to mitochondria and its loss causes arginine accumulation,which disrupts mitochondrial dynamics.Heterologous expression of human ARGl or ARG2 rescued the mitochondrial defects of argn-1 mutants.Importantly,genetic inactivation of the mitochondrial basic amino acid transporter SLC-25A29 or the mitochondrial glutamate transporter SLC-25A18.1 fully suppressed the mitochondrial defects caused by argn-1 mutations.These findings suggest that mitochondrial damage probably contributes to the pathogenesis of hyperargininemia and provide clues for developing therapeutic treatments for hyperargininemia.