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1.
Nat Commun ; 13(1): 6339, 2022 10 25.
Article in English | MEDLINE | ID: mdl-36284093

ABSTRACT

Twenty-nine years following the breakthrough discovery that a single-gene mutation of daf-2 doubles Caenorhabditis elegans lifespan, it remains unclear where this insulin/IGF-1 receptor gene is expressed and where it acts to regulate ageing. Using knock-in fluorescent reporters, we determined that daf-2 and its downstream transcription factor daf-16 are expressed ubiquitously. Using tissue-specific targeted protein degradation, we determined that intracellular DAF-2-to-DAF-16 signaling in the intestine plays a major role in lifespan regulation, while that in the hypodermis, neurons, and germline plays a minor role. Notably, intestine-specific loss of DAF-2 activates DAF-16 in and outside the intestine, causes almost no adverse effects on development and reproduction, and extends lifespan by 94% in a way that partly requires non-intestinal DAF-16. Consistent with intestine supplying nutrients to the entire body, evidence from this and other studies suggests that altered metabolism, particularly down-regulation of protein and RNA synthesis, mediates longevity by reduction of insulin/IGF-1 signaling.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Animals , Caenorhabditis elegans/metabolism , Longevity/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Insulin/metabolism , Mutation , Intestines , RNA/metabolism
2.
Sci China Life Sci ; 64(12): 2153-2174, 2021 12.
Article in English | MEDLINE | ID: mdl-34755252

ABSTRACT

Developmental diapause is a widespread strategy for animals to survive seasonal starvation and environmental harshness. Diapaused animals often ration body fat to generate a basal level of energy for enduring survival. How diapause and fat rationing are coupled, however, is poorly understood. The nematode Caenorhabditis elegans excretes pheromones to the environment to induce a diapause form called dauer larva. Through saturated forward genetic screens and CRISPR knockout, we found that dauer pheromones feed back to repress the transcription of ACOX-3, MAOC-1, DHS-28, DAF-22 (peroxisomal ß-oxidation enzymes dually involved in pheromone synthesis and fat burning), ALH-4 (aldehyde dehydrogenase for pheromone synthesis), PRX-10 and PRX-11 (peroxisome assembly and proliferation factors). Dysfunction of these pheromone enzymes and factors relieves the repression. Surprisingly, transcription is repressed not by pheromones excreted but by pheromones endogenous to each animal. The endogenous pheromones regulate the nuclear translocation of HNF4α family nuclear receptor NHR-79 and its co-receptor NHR-49, and, repress transcription through the two receptors. The feedback repression maintains pheromone homeostasis, increases fat storage, decreases fat burning, and prolongs dauer lifespan. Thus, the exocrine dauer pheromones possess an unexpected endocrine function to mediate a peroxisome-nucleus crosstalk, coupling dauer diapause to fat rationing.


Subject(s)
Acyl-CoA Oxidase/metabolism , Caenorhabditis elegans/metabolism , Fatty Acids/metabolism , Hepatocyte Nuclear Factor 4/metabolism , Pheromones/metabolism , Adipose Tissue/metabolism , Animals , Caenorhabditis elegans/genetics , Diapause/physiology , Homeostasis/physiology , Larva , Oxidation-Reduction , Peroxisomes/metabolism , Transcription, Genetic
3.
Proc Natl Acad Sci U S A ; 114(33): 8841-8846, 2017 08 15.
Article in English | MEDLINE | ID: mdl-28760992

ABSTRACT

Nuclear receptors play important roles in regulating fat metabolism and energy production in humans. The regulatory functions and endogenous ligands of many nuclear receptors are still unidentified, however. Here, we report that CYP-37A1 (ortholog of human cytochrome P450 CYP4V2), EMB-8 (ortholog of human P450 oxidoreductase POR), and DAF-12 (homolog of human nuclear receptors VDR/LXR) constitute a hormone synthesis and nuclear receptor pathway in Caenorhabditis elegans This pathway specifically regulates the thermosensitive fusion of fat-storing lipid droplets. CYP-37A1, together with EMB-8, synthesizes a lipophilic hormone not identical to Δ7-dafachronic acid, which represses the fusion-promoting function of DAF-12. CYP-37A1 also negatively regulates thermotolerance and lifespan at high temperature in a DAF-12-dependent manner. Human CYP4V2 can substitute for CYP-37A1 in C. elegans This finding suggests the existence of a conserved CYP4V2-POR-nuclear receptor pathway that functions in converting multilocular lipid droplets to unilocular ones in human cells; misregulation of this pathway may lead to pathogenic fat storage.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Cholestenes/metabolism , Cytochrome P-450 Enzyme System/metabolism , Hot Temperature , Lipid Droplets/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Cytochrome P-450 Enzyme System/genetics , Humans , Receptors, Cytoplasmic and Nuclear/genetics
4.
G3 (Bethesda) ; 6(8): 2407-19, 2016 08 09.
Article in English | MEDLINE | ID: mdl-27261001

ABSTRACT

To identify genes that regulate the dynamics of lipid droplet (LD) size, we have used the genetically tractable model organism Caenorhabditis elegans, whose wild-type LD population displays a steady state of size with an upper limit of 3 µm in diameter. From a saturated forward genetic screen of 6.7 × 10(5) mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 µm. These mutants define nine novel complementation groups, in addition to four known genes (maoc-1, dhs-28, daf-22, and prx-10). The nine groups are named drop (lipid droplet abnormal) and categorized into four classes. Class I mutants drop-5 and drop-9, similar to prx-10, are up-regulated in ACS-22-DGAT-2-dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2, drop-3, drop-6, and drop-7 are up-regulated in LD growth, are resistant to LD hydrolysis, but are not defective in peroxisome import. Class III mutants drop-1 and drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22-independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Lipid Droplets/metabolism , Lipid Metabolism/genetics , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/isolation & purification , Caenorhabditis elegans Proteins/metabolism , Genome , Haploidy , Mutation
5.
J Cell Biol ; 198(5): 895-911, 2012 Sep 03.
Article in English | MEDLINE | ID: mdl-22927462

ABSTRACT

At the subcellular level, fat storage is confined to the evolutionarily conserved compartments termed lipid droplets (LDs), which are closely associated with the endoplasmic reticulum (ER). However, the molecular mechanisms that enable ER-LD interaction and facilitate neutral lipid loading into LDs are poorly understood. In this paper, we present evidence that FATP1/acyl-CoA synthetase and DGAT2/diacylglycerol acyltransferase are components of a triglyceride synthesis complex that facilitates LD expansion. A loss of FATP1 or DGAT2 function blocked LD expansion in Caenorhabditis elegans. FATP1 preferentially associated with DGAT2, and they acted synergistically to promote LD expansion in mammalian cells. Live imaging indicated that FATP1 and DGAT2 are ER and LD resident proteins, respectively, and electron microscopy revealed FATP1 and DGAT2 foci close to the LD surface. Furthermore, DGAT2 that was retained in the ER failed to support LD expansion. We propose that the evolutionarily conserved FATP1-DGAT2 complex acts at the ER-LD interface and couples the synthesis and deposition of triglycerides into LDs both physically and functionally.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Diacylglycerol O-Acyltransferase/metabolism , Endoplasmic Reticulum/metabolism , Fatty Acid Transport Proteins/metabolism , Lipid Metabolism/physiology , Animals , Caenorhabditis elegans , Triglycerides/metabolism
6.
Mol Cell Proteomics ; 11(8): 317-28, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22493183

ABSTRACT

Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved across almost all species. Many metabolic syndromes are directly linked to the over-storage of neutral lipids in LDs. The study of LDs in Caenorhabditis elegans (C. elegans) has been difficult because of the lack of specific LD marker proteins. Here we report the purification and proteomic analysis of C. elegans lipid droplets for the first time. We identified 306 proteins, 63% of these proteins were previously known to be LD-proteins, suggesting a similarity between mammalian and C. elegans LDs. Using morphological and biochemical analyses, we show that short-chain dehydrogenase, DHS-3 is almost exclusively localized on C. elegans LDs, indicating that it can be used as a LD marker protein in C. elegans. These results will facilitate further mechanistic studies of LDs in this powerful genetic system, C. elegans.


Subject(s)
Biomarkers/analysis , Butyryl-CoA Dehydrogenase/analysis , Caenorhabditis elegans Proteins/analysis , Caenorhabditis elegans/metabolism , Cytoplasmic Vesicles/metabolism , Proteome/analysis , Proteomics/methods , Animals , Blotting, Western , Cytoplasmic Vesicles/ultrastructure , Lipid Metabolism , Lipids/chemistry , Mass Spectrometry , Microscopy, Confocal , Microscopy, Electron, Transmission
7.
BMC Cell Biol ; 11: 96, 2010 Dec 08.
Article in English | MEDLINE | ID: mdl-21143850

ABSTRACT

BACKGROUND: Lipid droplets are a class of eukaryotic cell organelles for storage of neutral fat such as triacylglycerol (TAG) and cholesterol ester (CE). We and others have recently reported that lysosome-related organelles (LROs) are not fat storage structures in the nematode C. elegans. We also reported the formation of enlarged lipid droplets in a class of peroxisomal fatty acid ß-oxidation mutants. In the present study, we seek to provide further evidence on the organelle nature and biophysical properties of fat storage structures in wild-type and mutant C. elegans. RESULTS: In this study, we provide biochemical, histological and ultrastructural evidence of lipid droplets in wild-type and mutant C. elegans that lack lysosome related organelles (LROs). The formation of lipid droplets and the targeting of BODIPY fatty acid analogs to lipid droplets in live animals are not dependent on lysosomal trafficking or peroxisome dysfunction. However, the targeting of Nile Red to lipid droplets in live animals occurs only in mutants with defective peroxisomes. Nile Red labelled-lipid droplets are characterized by a fluorescence emission spectrum distinct from that of Nile Red labelled-LROs. Moreover, we show that the recently developed post-fix Nile Red staining method labels lipid droplets exclusively. CONCLUSIONS: Our results demonstrate lipid droplets as ubiquitous fat storage organelles and provide a unified explanation for previous studies on fat labelling methods in C. elegans. These results have important applications to the studies of fat storage and lipid droplet regulation in the powerful genetic system, C. elegans.


Subject(s)
Caenorhabditis elegans/metabolism , Lipid Metabolism/physiology , Animals , Caenorhabditis elegans/ultrastructure , Cholesterol Esters/metabolism , Fluorescent Dyes/chemistry , Lysosomes/metabolism , Oxazines/chemistry , Peroxisomes/metabolism , Triglycerides/metabolism
8.
Proc Natl Acad Sci U S A ; 107(10): 4640-5, 2010 Mar 09.
Article in English | MEDLINE | ID: mdl-20176933

ABSTRACT

Dietary fat accumulates in lipid droplets or endolysosomal compartments that undergo selective expansion under normal or pathophysiological conditions. We find that genetic defects in a peroxisomal beta-oxidation pathway cause size expansion in lipid droplets that are distinct from the lysosome-related organelles in Caenorhabditis elegans. Expansion of lipid droplets is accompanied by an increase in triglycerides (TAG) that are resistant to fasting- or TAG lipase-triggered lipolysis. Nevertheless, in mutant animals, a diet poor in vaccenic acid reduced the TAG level and lipid droplet size. Our results implicate peroxisomal dysfunction in pathologic lipid droplet expansion in animals and illustrate how dietary factors modulate the phenotype of such genetic defects.


Subject(s)
Caenorhabditis elegans/metabolism , Cytoplasmic Granules/metabolism , Lipid Metabolism , Lipids/chemistry , Animals , Animals, Genetically Modified , Blotting, Western , Caenorhabditis elegans/genetics , Caenorhabditis elegans/ultrastructure , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cytoplasmic Granules/ultrastructure , Dietary Fats/administration & dosage , Dietary Fats/metabolism , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Lipase/genetics , Lipase/metabolism , Lipolysis , Lysosomes/metabolism , Male , Microscopy, Confocal , Microscopy, Electron , Mutation , Oleic Acids/administration & dosage , Oleic Acids/metabolism , Oxidation-Reduction , Peroxisomes/metabolism , Triglycerides/metabolism
9.
BMC Genomics ; 11: 13, 2010 Jan 07.
Article in English | MEDLINE | ID: mdl-20055996

ABSTRACT

BACKGROUND: Considerable progress has been made in our understanding of sex determination and dosage compensation mechanisms in model organisms such as C. elegans, Drosophila and M. musculus. Strikingly, the mechanism involved in sex determination and dosage compensation are very different among these three model organisms. Birds present yet another situation where the heterogametic sex is the female. Sex determination is still poorly understood in birds and few key determinants have so far been identified. In contrast to most other species, dosage compensation of bird sex chromosomal genes appears rather ineffective. RESULTS: By comparing microarrays from microdissected primitive streak from single chicken embryos, we identified a large number of genes differentially expressed between male and female embryos at a very early stage (Hamburger and Hamilton stage 4), long before any sexual differentiation occurs. Most of these genes are located on the Z chromosome, which indicates that dosage compensation is ineffective in early chicken embryos. Gene ontology analyses, using an enhanced annotation tool for Affymetrix probesets of the chicken genome developed in our laboratory (called Manteia), show that among these male-biased genes found on the Z chromosome, more than 20 genes play a role in sex differentiation. CONCLUSIONS: These results corroborate previous studies demonstrating the rather inefficient dosage compensation for Z chromosome in birds and show that this sexual dimorphism in gene regulation is observed long before the onset of sexual differentiation. These data also suggest a potential role of non-compensated Z-linked genes in somatic sex differentiation in birds.


Subject(s)
Chick Embryo/embryology , Dosage Compensation, Genetic , Gene Expression Regulation, Developmental , Sex Characteristics , Animals , Female , Gastrulation , Gene Expression Profiling , Male , Oligonucleotide Array Sequence Analysis , Sex Chromosomes/genetics
10.
Dev Biol ; 336(1): 112-21, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19747476

ABSTRACT

Embryonic segmentation in clitellate annelids (oligochaetes and leeches) is a cell lineage-driven process. Embryos of these worms generate a posterior growth zone consisting of 5 bilateral pairs of identified segmentation stem cells (teloblasts), each of which produces a column of segmental founder cells (blast cells). Each blast cell generates a lineage-specific clone via a stereotyped sequence of cell divisions, which are typically unequal both in terms of the relative size of the sister cells and in the progeny to which they give rise. In two of the five teloblast lineages, including the ventralmost, primary neurogenic (N) lineage, the blast cells adopt two different fates, designated nf and ns, in exact alternation within the blast cell column; this is termed a grandparental stem cell lineage. To lay groundwork for investigating unequal divisions in the leech Helobdella, we have surveyed the Helobdella robusta genome for genes encoding orthologs of the Rho family GTPases, including the rho, rac and cdc42 sub-families, which are known to be involved in multiple processes involving cell polarization in other systems. We find that, in contrast to most other known systems the Helobdella genome contains two cdc42 orthologs, one of which is expressed at higher levels in the ns blast cells than in nf blast cells. We also demonstrate that the asymmetric divisions of the primary nf and ns blast cells are regulated by the polarized distribution of the activated form of the Cdc42 protein, rather than by the overall level of expression. Our results provide the first molecular insights into the mechanisms of the grandparental stem cell lineages, a novel, yet evolutionarily ancient stem cell division pattern. Our results also provide an example in which asymmetries in the distribution of Cdc42 activity, rather than in the overall levels of Cdc42 protein, are important regulating unequal divisions in animal cells.


Subject(s)
Cell Lineage , Embryo, Nonmammalian/cytology , Leeches/cytology , Stem Cells/cytology , Animals , Body Patterning/genetics , Cell Differentiation , Cloning, Molecular , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Immunohistochemistry , In Situ Hybridization , Leeches/embryology , Leeches/genetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Confocal , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Stem Cells/metabolism , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolism , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism , RAC2 GTP-Binding Protein
11.
Development ; 132(9): 2103-13, 2005 May.
Article in English | MEDLINE | ID: mdl-15788451

ABSTRACT

Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos. We have applied them to the primary neurogenic (N) lineage, in which alternate segmental founder cells (nf and ns blast cells) contribute distinct sets of progeny to the segmental ganglia. The nf and ns blast cell clones exhibit strikingly different cell division patterns: the increase in cell number within the nf clone is roughly linear, while that in the ns clone is almost exponential. To analyze spindle dynamics in the asymmetric divisions of individual blast cells, we have injected teloblasts with mRNA encoding a tau::GFP fusion protein. Our results show that the asymmetric divisions of n blast cells result from a posterior shift of both the spindle within the cell and the midbody within the mitotic spindle, with differential regulation of these processes between nf and ns.


Subject(s)
Body Patterning/physiology , Cell Division/physiology , Cell Lineage/physiology , Leeches/embryology , RNA, Messenger/metabolism , Animals , Biomarkers , Genes, Reporter , Leeches/cytology , Mitosis/physiology , RNA, Messenger/administration & dosage , Spindle Apparatus/metabolism
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