Lipid droplets as ubiquitous fat storage organelles in C. elegans

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 β-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.Peer Reviewe

Metabolic Impact of Adult-Onset, Isolated, Growth Hormone Deficiency (AOiGHD) Due to Destruction of Pituitary Somatotropes

Growth hormone (GH) inhibits fat accumulation and promotes protein accretion, therefore the fall in GH observed with weight gain and normal aging may contribute to metabolic dysfunction. To directly test this hypothesis a novel mouse model of adult onset-isolated GH deficiency (AOiGHD) was generated by cross breeding rat GH promoter-driven Cre recombinase mice (Cre) with inducible diphtheria toxin receptor mice (iDTR) and treating adult Cre+/−,iDTR+/− offspring with DT to selectively destroy the somatotrope population of the anterior pituitary gland, leading to a reduction in circulating GH and IGF-I levels. DT-treated Cre−/−,iDTR+/− mice were used as GH-intact controls. AOiGHD improved whole body insulin sensitivity in both low-fat and high-fat fed mice. Consistent with improved insulin sensitivity, indirect calorimetry revealed AOiGHD mice preferentially utilized carbohydrates for energy metabolism, as compared to GH-intact controls. In high-fat, but not low-fat fed AOiGHD mice, fat mass increased, hepatic lipids decreased and glucose clearance and insulin output were impaired. These results suggest the age-related decline in GH helps to preserve systemic insulin sensitivity, and in the context of moderate caloric intake, prevents the deterioration in metabolic function. However, in the context of excess caloric intake, low GH leads to impaired insulin output, and thereby could contribute to the development of diabetes

The SMC Loader Scc2 Promotes ncRNA Biogenesis and Translational Fidelity

International audienceThe Scc2-Scc4 complex is essential for loading the cohesin complex onto DNA. Cohesin has important roles in chromosome segregation, DSB repair, and chromosome condensation. Here we report that Scc2 is important for gene expression in budding yeast. Scc2 and the transcriptional regulator Paf1 collaborate to promote the production of Box H/ACA snoR-NAs which guide pseudouridylation of RNAs including ribosomal RNA. Mutation of SCC2 was associated with defects in the production of ribosomal RNA, ribosome assembly, and splicing. While the scc2 mutant does not have a general defect in protein synthesis, it shows increased frameshifting and reduced cap-independent translation. These findings suggest Scc2 normally promotes a gene expression program that supports translational fidelity. We hypothesize that translational dysfunction may contribute to the human disorder Cornelia de Lange syndrome, which is caused by mutations in NIPBL, the human ortholog of SCC2

Sns and Kirre, the Drosophila orthologs of Nephrin and Neph1, direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytes

The Immunoglobulin superfamily (IgSF) proteins Neph1 and Nephrin are co-expressed within podocytes in the kidney glomerulus, where they localize to the slit diaphragm (SD) and contribute to filtration between blood and urine. Herein, we demonstrate that their Drosophila orthologs Kirre (Duf) and Sns are co-expressed within binucleate garland cell nephrocytes (GCNs) that contribute to detoxification of the insect hemolymph by uptake of molecules through an SD-like nephrocyte diaphragm (ND) into labyrinthine channels that are active sites of endocytosis. The functions of Kirre and Sns in the embryonic musculature, to mediate adhesion and fusion between myoblasts to form multinucleate muscle fibers, have been conserved in the GCNs, where they contribute to adhesion of GCNs in the `garland' and to their fusion into binucleate cells. Sns and Kirre proteins localize to the ND at the entry point into the labyrinthine channels and, like their vertebrate counterparts, are essential for its formation. Knockdown of Kirre or Sns drastically reduces the number of NDs at the cell surface. These defects are associated with a decrease in uptake of large proteins, suggesting that the ND distinguishes molecules of different sizes and controls access to the channels. Moreover, mutations in the Sns fibronectin-binding or immunoglobulin domains lead to morphologically abnormal NDs and to reduced passage of proteins into the labyrinthine channels for uptake by endocytosis, suggesting a crucial and direct role for Sns in ND formation and function. These data reveal significant similarities between the insect ND and the SD in mammalian podocytes at the level of structure and function

<i>scc2-4</i> is a partial loss of function mutation.

<p>(A) Scc2 has conserved HEAT repeats in the C terminal domain. (B) The amino acid mutated in <i>scc2-4</i> is conserved from yeast to human (E534). (C) A growth curve of the WT and <i>scc2-4</i> mutant strains in YPD at 30°C is shown (left). Growth curves were measured using a TECAN machine. The <i>scc2-4</i> mutant has a slower maximum growth rate at 30°C compared to the WT, p = 0.0001 (right). (D) The levels of Scc2-Myc were measured by Western blotting (left) and quantified (right, in triplicate with error bars). Pgk1 serves as a loading control. The E534K mutation does not significantly reduce the amount of Scc2 protein at permissive temperature (30°C).</p

Translational fidelity is reduced in the <i>scc2-4</i> mutant.

<p>Dual luciferase reporters were used to measure translation. (A, top) Schematic diagram of CrPV IGR IRES containing reporter. Renilla luciferase is translated by a cap-dependent mechanism, and firefly luciferase synthesis requires cap-independent initiation mediated by the IRES. While cap-dependent translation is mildly impaired (p = 0.01), IRES-dependent translation is more strongly inhibited in the <i>scc2-4</i> mutant (p = 0.001). (B, top) A dual luciferase reporter was used to monitor -1 frameshifting mediated by sequence derived from the yeast L-A virus, and +1 frameshifting promoted by the yeast Ty1 sequence. In-frame renilla luciferase translation serves as a normalization control, and efficiencies were determined as previously described (Landry et al., 2009) The mutant shows an increase in -1 frameshifting. (C). Readthrough efficiency for three different stop codons (UAA, UAG, UGA) was measured for the WT and <i>scc2-4</i> strains. The translation of renilla luciferase serves as a normalization control. (D) The maximal growth rates of the WT and <i>scc2-4</i> mutant strains were compared, with the WT growth rate set to 100%. Strains were grown in YPD or YPD with paromomycin. p-values were calculated using student two-tailed t-test (asterisk indicates p<0.05). Standard error is indicated for at least three independent measurements.</p

Scc2 and Paf1 recruitment at snoDNAs is co-dependent.

<p>(A) WT, <i>scc2-4</i>, and <i>paf1Δ</i> mutant strains were cultured in YPD medium to mid-log phase (~ OD₆₀₀ = 0.5–0.8). Strains were crosslinked and chromatin extracted for ChIP. ChIP/qPCR analysis was carried out for Scc2-Myc and Scc2^E534K-Myc and Scc2-Myc <i>paf1Δ</i>. ChIP performed without the addition of primary antibody served as a negative control. ChIP experiments were performed at least three times. p-values were calculated using a student t-test. Standard error bars are indicated for n = 3. Values different from the WT are indicated by an asterisk (p<0.05). qPCR analysis shows reduced enrichment of indicated snoDNAs in mutant relative to WT in Paf1 ChIP (α-HA antibody, 12CA5, Roche). (B) Western blot analysis shows that the protein level of Paf1 is reduced in the <i>scc2-4</i> mutant. Scc2 levels also appear reduced in the <i>paf1Δ</i> strain. Pgk1 served as the loading control. (C) Western blot analysis shows that the protein level of Ctr9 is reduced in the <i>scc2-4</i> mutant. (D) qPCR analysis shows reduced enrichment of snoDNAs in the Scc2-Myc ChIP in the <i>paf1Δ</i> mutant strain relative to WT (α-Myc antibody, 9B11, Cell signaling). (E) A cartoon model for the co-recruitment of Scc2 and Paf1 at snoDNAs is shown. The Tbf1 transcription factor is shown in purple, RNA Pol II in yellow, and Scc2 and Paf1c in blue and green, respectively.</p

The <i>scc2-4</i> mutation compromises the association with genic regions at 30°C.

<p>WT and <i>scc2-4</i> mutant strains were cultured to mid-log phase (~OD₆₀₀ = 0.5–0.8) in YPD medium. Strains were crosslinked and chromatin extracted for ChIP. Metagene analysis was carried out for Scc2-Myc and Scc2^E534K-Myc for ChIP seq data. Two biological replicates for each are shown. (A) The <i>scc2-4</i> mutation does not affect the association of Scc2 with centromere regions. (B) The mutation reduces the association with ribosomal protein genes (132) (C) rDNA (D) snoDNAs (77) and (E) tDNAs (275).</p

Hundreds of genes are differentially expressed in the <i>scc2-4</i> mutant compared to WT at 30°C in YPD.

<p>(A) Gene expression values (mean of triplicate samples) are shown in MA plot. Differentially expressed genes (adjusted p-value <0.05) were colored in orange, and then colored red or green after a minimum fold change cut-off of 1.5 was applied. There are 2644 genes differentially expressed genes in the <i>scc2-4</i> mutant, with 1285 up-regulated and 1359 down-regulated. Applying a fold-change cutoff of 1.5 (corresponding to an absolute log₂ value of 0.6) returns a more reasonable number of genes for GO analysis and general comparison. (B) GO term analysis for the up-regulated genes shows enrichment for genes important for RNA processing/metabolism and ribosome biogenesis. (C) GO term analysis for down-regulated genes shows enrichment for genes important for biological processes such as oxidative phosphorylation, electron transport chain, and carbohydrate metabolic processes.</p