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The Causative Gene in Chanarian Dorfman Syndrome Regulates Lipid Droplet Homeostasis in C. elegans.

Xie M, Roy R - PLoS Genet. (2015)

Bottom Line: We found that the compromise of one of the three C. elegans orthologues of human cgi-58 significantly improves the survival of AMPK-deficient dauers.We also provide evidence that C. elegans CGI-58 acts as a co-activator of ATGL-1, while it also functions cooperatively to maintain regular lipid droplet structure.Surprisingly, we show that it also acts independently of ATGL-1 to restrict lipid droplet coalescence by altering the surface abundance and composition of long chain (C20) polyunsaturated fatty acids (PUFAs).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, Penfield, Montreal, Quebec, Canada.

ABSTRACT
AMP-activated kinase (AMPK) is a key regulator of many cellular mechanisms required for adjustment to various stresses induced by the changing environment. In C. elegans dauer larvae AMPK- mutants expire prematurely due to hyperactive Adipose Triglyceride Lipase (ATGL-1) followed by rapid depletion of triglyceride stores. We found that the compromise of one of the three C. elegans orthologues of human cgi-58 significantly improves the survival of AMPK-deficient dauers. We also provide evidence that C. elegans CGI-58 acts as a co-activator of ATGL-1, while it also functions cooperatively to maintain regular lipid droplet structure. Surprisingly, we show that it also acts independently of ATGL-1 to restrict lipid droplet coalescence by altering the surface abundance and composition of long chain (C20) polyunsaturated fatty acids (PUFAs). Our data reveal a novel structural role of CGI-58 in maintaining lipid droplet homeostasis through its effects on droplet composition, morphology and lipid hydrolysis; a conserved function that may account for some of the ATGL-1-independent features unique to Chanarin-Dorfman Syndrome.

No MeSH data available.


Related in: MedlinePlus

CGI-58 interacts with ATGL-1 and tethers it to the lipid droplets.(A) CGI-58::GFP was expressed in the hypodermis and the intestine at comparable levels in both control daf-2 and daf-2; aak(0) mutant dauer larvae. Both strains carry the same ex[Pcgi-58::cgi-58::GFP; rol-6(gf)] transgene (See Materials and Methods). Scale bar = 20 μm. (B) CGI-58::GFP localizes to the surface of the lipid droplets at dauer day 0 (48 hours after being shifted to 25°C at the L1 stage) in both daf-2 and daf-2; aak(0) mutant dauers. Both strains carry the same ex[Pcgi-58::cgi-58::GFP; rol-6(gf)] transgene. Scale bar = 10μm. Insets (B' and B") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same magnification. (C) Optimal ATGL-1 lipase activity requires cgi-58. ATGL-1-dependent lipase activity was determined in daf-2; aak(0) mutant dauer larvae with wild type or compromised cgi-58/atgl-1 function. ** indicates statistical significance (P<0.01) compared to all three of the other genotypes. Error bars indicate SD of three independent experiments. (D) Elimination of cgi-58 resulted in the dissociation of ATGL-1 from the lipid droplets. Both strains carry the same hjIs67[Patgl-1::atgl-1::GFP] transgene. Scale bar = 10μm. Insets (D' and D") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same final magnification. (E) ATGL-1 association with the lipid droplets is dependent on appropriate CGI-58 levels. Immunoblot analysis was used to determine the levels of ATGL-1 in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 dauer extracts of each genotype. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level. (F) CGI-58 does not contribute to ATGL-1 stability in AMPK mutant dauers. ATGL-1 levels were determined by immunobloting using anti-ATGL-1 antisera in lysates obtained from AMPK mutants with or without cgi-58. (G) CGI-58 physically interacts with ATGL-1 in vivo in both control and AMPK mutant dauers. Co-immunoprecipitations were performed with daf-2 and daf-2; aak(0) day 0 dauer larvae carrying the same ex[Pcgi-58::cgi-58::GFP;rol-6(gf)] transgene using anti-ATGL-1 serum for pull down and blotted with anti-GFP serum.
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pgen.1005284.g002: CGI-58 interacts with ATGL-1 and tethers it to the lipid droplets.(A) CGI-58::GFP was expressed in the hypodermis and the intestine at comparable levels in both control daf-2 and daf-2; aak(0) mutant dauer larvae. Both strains carry the same ex[Pcgi-58::cgi-58::GFP; rol-6(gf)] transgene (See Materials and Methods). Scale bar = 20 μm. (B) CGI-58::GFP localizes to the surface of the lipid droplets at dauer day 0 (48 hours after being shifted to 25°C at the L1 stage) in both daf-2 and daf-2; aak(0) mutant dauers. Both strains carry the same ex[Pcgi-58::cgi-58::GFP; rol-6(gf)] transgene. Scale bar = 10μm. Insets (B' and B") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same magnification. (C) Optimal ATGL-1 lipase activity requires cgi-58. ATGL-1-dependent lipase activity was determined in daf-2; aak(0) mutant dauer larvae with wild type or compromised cgi-58/atgl-1 function. ** indicates statistical significance (P<0.01) compared to all three of the other genotypes. Error bars indicate SD of three independent experiments. (D) Elimination of cgi-58 resulted in the dissociation of ATGL-1 from the lipid droplets. Both strains carry the same hjIs67[Patgl-1::atgl-1::GFP] transgene. Scale bar = 10μm. Insets (D' and D") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same final magnification. (E) ATGL-1 association with the lipid droplets is dependent on appropriate CGI-58 levels. Immunoblot analysis was used to determine the levels of ATGL-1 in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 dauer extracts of each genotype. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level. (F) CGI-58 does not contribute to ATGL-1 stability in AMPK mutant dauers. ATGL-1 levels were determined by immunobloting using anti-ATGL-1 antisera in lysates obtained from AMPK mutants with or without cgi-58. (G) CGI-58 physically interacts with ATGL-1 in vivo in both control and AMPK mutant dauers. Co-immunoprecipitations were performed with daf-2 and daf-2; aak(0) day 0 dauer larvae carrying the same ex[Pcgi-58::cgi-58::GFP;rol-6(gf)] transgene using anti-ATGL-1 serum for pull down and blotted with anti-GFP serum.

Mentions: To better understand the role of C. elegans CGI-58 in regulating ATGL-1, we generated a CGI-58::GFP translational fusion transgene and introduced it into control daf-2 animals (daf-2 dauer larvae will hitherto be referred to as control dauers) that were AMPK-deficient. During the dauer stage, C. elegans CGI-58 is expressed in both control and AMPK mutant dauer larvae mainly in the hypodermis and intestine (Fig 2A), the two tissues that have been well characterized for their role in lipid synthesis and storage. Next, we stained the lipid droplets of these animals with red C1-BODIPY-C12 and found that most of the GFP-tagged C. elegans CGI-58 proteins are closely associated with the lipid droplets in both control daf-2 and AMPK-deficient dauer larvae (B' and B" in Fig 2B).


The Causative Gene in Chanarian Dorfman Syndrome Regulates Lipid Droplet Homeostasis in C. elegans.

Xie M, Roy R - PLoS Genet. (2015)

CGI-58 interacts with ATGL-1 and tethers it to the lipid droplets.(A) CGI-58::GFP was expressed in the hypodermis and the intestine at comparable levels in both control daf-2 and daf-2; aak(0) mutant dauer larvae. Both strains carry the same ex[Pcgi-58::cgi-58::GFP; rol-6(gf)] transgene (See Materials and Methods). Scale bar = 20 μm. (B) CGI-58::GFP localizes to the surface of the lipid droplets at dauer day 0 (48 hours after being shifted to 25°C at the L1 stage) in both daf-2 and daf-2; aak(0) mutant dauers. Both strains carry the same ex[Pcgi-58::cgi-58::GFP; rol-6(gf)] transgene. Scale bar = 10μm. Insets (B' and B") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same magnification. (C) Optimal ATGL-1 lipase activity requires cgi-58. ATGL-1-dependent lipase activity was determined in daf-2; aak(0) mutant dauer larvae with wild type or compromised cgi-58/atgl-1 function. ** indicates statistical significance (P<0.01) compared to all three of the other genotypes. Error bars indicate SD of three independent experiments. (D) Elimination of cgi-58 resulted in the dissociation of ATGL-1 from the lipid droplets. Both strains carry the same hjIs67[Patgl-1::atgl-1::GFP] transgene. Scale bar = 10μm. Insets (D' and D") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same final magnification. (E) ATGL-1 association with the lipid droplets is dependent on appropriate CGI-58 levels. Immunoblot analysis was used to determine the levels of ATGL-1 in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 dauer extracts of each genotype. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level. (F) CGI-58 does not contribute to ATGL-1 stability in AMPK mutant dauers. ATGL-1 levels were determined by immunobloting using anti-ATGL-1 antisera in lysates obtained from AMPK mutants with or without cgi-58. (G) CGI-58 physically interacts with ATGL-1 in vivo in both control and AMPK mutant dauers. Co-immunoprecipitations were performed with daf-2 and daf-2; aak(0) day 0 dauer larvae carrying the same ex[Pcgi-58::cgi-58::GFP;rol-6(gf)] transgene using anti-ATGL-1 serum for pull down and blotted with anti-GFP serum.
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Related In: Results  -  Collection

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pgen.1005284.g002: CGI-58 interacts with ATGL-1 and tethers it to the lipid droplets.(A) CGI-58::GFP was expressed in the hypodermis and the intestine at comparable levels in both control daf-2 and daf-2; aak(0) mutant dauer larvae. Both strains carry the same ex[Pcgi-58::cgi-58::GFP; rol-6(gf)] transgene (See Materials and Methods). Scale bar = 20 μm. (B) CGI-58::GFP localizes to the surface of the lipid droplets at dauer day 0 (48 hours after being shifted to 25°C at the L1 stage) in both daf-2 and daf-2; aak(0) mutant dauers. Both strains carry the same ex[Pcgi-58::cgi-58::GFP; rol-6(gf)] transgene. Scale bar = 10μm. Insets (B' and B") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same magnification. (C) Optimal ATGL-1 lipase activity requires cgi-58. ATGL-1-dependent lipase activity was determined in daf-2; aak(0) mutant dauer larvae with wild type or compromised cgi-58/atgl-1 function. ** indicates statistical significance (P<0.01) compared to all three of the other genotypes. Error bars indicate SD of three independent experiments. (D) Elimination of cgi-58 resulted in the dissociation of ATGL-1 from the lipid droplets. Both strains carry the same hjIs67[Patgl-1::atgl-1::GFP] transgene. Scale bar = 10μm. Insets (D' and D") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same final magnification. (E) ATGL-1 association with the lipid droplets is dependent on appropriate CGI-58 levels. Immunoblot analysis was used to determine the levels of ATGL-1 in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 dauer extracts of each genotype. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level. (F) CGI-58 does not contribute to ATGL-1 stability in AMPK mutant dauers. ATGL-1 levels were determined by immunobloting using anti-ATGL-1 antisera in lysates obtained from AMPK mutants with or without cgi-58. (G) CGI-58 physically interacts with ATGL-1 in vivo in both control and AMPK mutant dauers. Co-immunoprecipitations were performed with daf-2 and daf-2; aak(0) day 0 dauer larvae carrying the same ex[Pcgi-58::cgi-58::GFP;rol-6(gf)] transgene using anti-ATGL-1 serum for pull down and blotted with anti-GFP serum.
Mentions: To better understand the role of C. elegans CGI-58 in regulating ATGL-1, we generated a CGI-58::GFP translational fusion transgene and introduced it into control daf-2 animals (daf-2 dauer larvae will hitherto be referred to as control dauers) that were AMPK-deficient. During the dauer stage, C. elegans CGI-58 is expressed in both control and AMPK mutant dauer larvae mainly in the hypodermis and intestine (Fig 2A), the two tissues that have been well characterized for their role in lipid synthesis and storage. Next, we stained the lipid droplets of these animals with red C1-BODIPY-C12 and found that most of the GFP-tagged C. elegans CGI-58 proteins are closely associated with the lipid droplets in both control daf-2 and AMPK-deficient dauer larvae (B' and B" in Fig 2B).

Bottom Line: We found that the compromise of one of the three C. elegans orthologues of human cgi-58 significantly improves the survival of AMPK-deficient dauers.We also provide evidence that C. elegans CGI-58 acts as a co-activator of ATGL-1, while it also functions cooperatively to maintain regular lipid droplet structure.Surprisingly, we show that it also acts independently of ATGL-1 to restrict lipid droplet coalescence by altering the surface abundance and composition of long chain (C20) polyunsaturated fatty acids (PUFAs).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, Penfield, Montreal, Quebec, Canada.

ABSTRACT
AMP-activated kinase (AMPK) is a key regulator of many cellular mechanisms required for adjustment to various stresses induced by the changing environment. In C. elegans dauer larvae AMPK- mutants expire prematurely due to hyperactive Adipose Triglyceride Lipase (ATGL-1) followed by rapid depletion of triglyceride stores. We found that the compromise of one of the three C. elegans orthologues of human cgi-58 significantly improves the survival of AMPK-deficient dauers. We also provide evidence that C. elegans CGI-58 acts as a co-activator of ATGL-1, while it also functions cooperatively to maintain regular lipid droplet structure. Surprisingly, we show that it also acts independently of ATGL-1 to restrict lipid droplet coalescence by altering the surface abundance and composition of long chain (C20) polyunsaturated fatty acids (PUFAs). Our data reveal a novel structural role of CGI-58 in maintaining lipid droplet homeostasis through its effects on droplet composition, morphology and lipid hydrolysis; a conserved function that may account for some of the ATGL-1-independent features unique to Chanarin-Dorfman Syndrome.

No MeSH data available.


Related in: MedlinePlus