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Perilipin-related protein regulates lipid metabolism in C. elegans.

Chughtai AA, Kaššák F, Kostrouchová M, Novotný JP, Krause MW, Saudek V, Kostrouch Z, Kostrouchová M - PeerJ (2015)

Bottom Line: Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes.In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals.Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague , Albertov, Prague , Czech Republic.

ABSTRACT
Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.

No MeSH data available.


Related in: MedlinePlus

The expression W01A8.1::gfp reporter genes in transgenic strains.W01A8.1a/c::GFP is shown in (A, C, E, G), and (I), and corresponding areas in Nomarski optics are shown in (B, D, F, H) and (J). (A) The onset of expression of W01A8.1a/c::GFP in epidermal cells and in intestinal cells of three-fold embryo. (C) The expression of W01A8.1a/c::GFP in intestinal cells of an L2 larva. (E) and (G) W01A8.1a/c::GFP expression in epidermal cells and intestinal cells of a young adult hermaphrodite. (G) GFP fluorescence around lipid droplet-like structures in the intestine that are marked by arrows and arrowheads. Corresponding image in Nomarski optics is in (H). (I) A higher magnification the lipid droplet-like structures in epidermal cells labeled by W01A8.1a/c::GFP (shown in Nomarski optics in the J). (K) Lipid droplets of an unfixed intestine labeled by W01A8.1b::GFP (corresponding Nomarski image is in L). (M, N) and (O) Part of the intestine of an adult larva expressing W01A8.1b::GFP (M) with corresponding staining of lipid droplets by LipidTox (N). (O) LipidTox-positive lipid droplets (red) with W01A8.1b::GFP on the periphery (green) in this merged view. Bars represent 50 µm in (B, H, J, L) and (N) and 100 µm in (D) and (F).
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fig-2: The expression W01A8.1::gfp reporter genes in transgenic strains.W01A8.1a/c::GFP is shown in (A, C, E, G), and (I), and corresponding areas in Nomarski optics are shown in (B, D, F, H) and (J). (A) The onset of expression of W01A8.1a/c::GFP in epidermal cells and in intestinal cells of three-fold embryo. (C) The expression of W01A8.1a/c::GFP in intestinal cells of an L2 larva. (E) and (G) W01A8.1a/c::GFP expression in epidermal cells and intestinal cells of a young adult hermaphrodite. (G) GFP fluorescence around lipid droplet-like structures in the intestine that are marked by arrows and arrowheads. Corresponding image in Nomarski optics is in (H). (I) A higher magnification the lipid droplet-like structures in epidermal cells labeled by W01A8.1a/c::GFP (shown in Nomarski optics in the J). (K) Lipid droplets of an unfixed intestine labeled by W01A8.1b::GFP (corresponding Nomarski image is in L). (M, N) and (O) Part of the intestine of an adult larva expressing W01A8.1b::GFP (M) with corresponding staining of lipid droplets by LipidTox (N). (O) LipidTox-positive lipid droplets (red) with W01A8.1b::GFP on the periphery (green) in this merged view. Bars represent 50 µm in (B, H, J, L) and (N) and 100 µm in (D) and (F).

Mentions: If the proteins encoded by W01A8.1 act as perilipins, they would be expected to be associated with lipid droplets (Kozusko et al., 2015). To test this, we created translational reporter transgenes regulated by the putative endogenous promoter expressing isoform b and lines in which the genomic locus was tagged by an in-frame C-terminal GFP cassette. The second transgene, W01A8.1a/c::gfp, is likely to express not only high levels of a and c tagged isoforms, but also the native isoform b (Fig. S1). The translational fusion constructs resulted in high levels of cytoplasmic proteins present in intestinal and epidermal cells on vesicular structures with the characteristic appearance of lipid droplets. This pattern of expression and cellular distribution was observed beginning at the three-fold embryonic stage and continued throughout development to adulthood (Fig. 2). To confirm that the observed GFP-associated vesicular structures were indeed lipid droplets, transgenic animals were stained with the lipophilic reagent LipidTox as previously described (O’Rourke et al., 2009). The translational GFP fusion protein reporters were localized at the periphery of fat droplets that were LipidTox positive (Fig. 2).


Perilipin-related protein regulates lipid metabolism in C. elegans.

Chughtai AA, Kaššák F, Kostrouchová M, Novotný JP, Krause MW, Saudek V, Kostrouch Z, Kostrouchová M - PeerJ (2015)

The expression W01A8.1::gfp reporter genes in transgenic strains.W01A8.1a/c::GFP is shown in (A, C, E, G), and (I), and corresponding areas in Nomarski optics are shown in (B, D, F, H) and (J). (A) The onset of expression of W01A8.1a/c::GFP in epidermal cells and in intestinal cells of three-fold embryo. (C) The expression of W01A8.1a/c::GFP in intestinal cells of an L2 larva. (E) and (G) W01A8.1a/c::GFP expression in epidermal cells and intestinal cells of a young adult hermaphrodite. (G) GFP fluorescence around lipid droplet-like structures in the intestine that are marked by arrows and arrowheads. Corresponding image in Nomarski optics is in (H). (I) A higher magnification the lipid droplet-like structures in epidermal cells labeled by W01A8.1a/c::GFP (shown in Nomarski optics in the J). (K) Lipid droplets of an unfixed intestine labeled by W01A8.1b::GFP (corresponding Nomarski image is in L). (M, N) and (O) Part of the intestine of an adult larva expressing W01A8.1b::GFP (M) with corresponding staining of lipid droplets by LipidTox (N). (O) LipidTox-positive lipid droplets (red) with W01A8.1b::GFP on the periphery (green) in this merged view. Bars represent 50 µm in (B, H, J, L) and (N) and 100 µm in (D) and (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562238&req=5

fig-2: The expression W01A8.1::gfp reporter genes in transgenic strains.W01A8.1a/c::GFP is shown in (A, C, E, G), and (I), and corresponding areas in Nomarski optics are shown in (B, D, F, H) and (J). (A) The onset of expression of W01A8.1a/c::GFP in epidermal cells and in intestinal cells of three-fold embryo. (C) The expression of W01A8.1a/c::GFP in intestinal cells of an L2 larva. (E) and (G) W01A8.1a/c::GFP expression in epidermal cells and intestinal cells of a young adult hermaphrodite. (G) GFP fluorescence around lipid droplet-like structures in the intestine that are marked by arrows and arrowheads. Corresponding image in Nomarski optics is in (H). (I) A higher magnification the lipid droplet-like structures in epidermal cells labeled by W01A8.1a/c::GFP (shown in Nomarski optics in the J). (K) Lipid droplets of an unfixed intestine labeled by W01A8.1b::GFP (corresponding Nomarski image is in L). (M, N) and (O) Part of the intestine of an adult larva expressing W01A8.1b::GFP (M) with corresponding staining of lipid droplets by LipidTox (N). (O) LipidTox-positive lipid droplets (red) with W01A8.1b::GFP on the periphery (green) in this merged view. Bars represent 50 µm in (B, H, J, L) and (N) and 100 µm in (D) and (F).
Mentions: If the proteins encoded by W01A8.1 act as perilipins, they would be expected to be associated with lipid droplets (Kozusko et al., 2015). To test this, we created translational reporter transgenes regulated by the putative endogenous promoter expressing isoform b and lines in which the genomic locus was tagged by an in-frame C-terminal GFP cassette. The second transgene, W01A8.1a/c::gfp, is likely to express not only high levels of a and c tagged isoforms, but also the native isoform b (Fig. S1). The translational fusion constructs resulted in high levels of cytoplasmic proteins present in intestinal and epidermal cells on vesicular structures with the characteristic appearance of lipid droplets. This pattern of expression and cellular distribution was observed beginning at the three-fold embryonic stage and continued throughout development to adulthood (Fig. 2). To confirm that the observed GFP-associated vesicular structures were indeed lipid droplets, transgenic animals were stained with the lipophilic reagent LipidTox as previously described (O’Rourke et al., 2009). The translational GFP fusion protein reporters were localized at the periphery of fat droplets that were LipidTox positive (Fig. 2).

Bottom Line: Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes.In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals.Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague , Albertov, Prague , Czech Republic.

ABSTRACT
Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.

No MeSH data available.


Related in: MedlinePlus