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A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance.

Pol A, Luetterforst R, Lindsay M, Heino S, Ikonen E, Parton RG - J. Cell Biol. (2001)

Bottom Line: The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis.Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets.We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, Centre for Microscopy and Microanalysis and Department of Physiology and Pharmacology, University of Queensland, Queensland 4072, Australia.

ABSTRACT
Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV), specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

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The cavDGV phenotype is a feature of all caveolin family members. a–c, BHK cells were cotransfected with full-length VSV-G–tagged cav-1 and HA-tagged cavDGV, and after 24 h were labeled with a rabbit antibody to VSV-G tag and a mouse antibody to HA tag. In a low but significant proportion of transfected cells (2–5% of transfectants) full-length cav-1 (green) accumulated in the same intracellular rings as cavDGV (red). Although in those cells, cav-1 was detected on the PM (a and c, arrowheads) cavDGV was totally excluded from the PM. Some ring-like structures were strongly labeled for full-length cav-1 but not for cavDGV (b and c arrows). d and e, BHK cells were transfected with HA-tagged cav-3DGV equivalent truncation mutants of cav-1 (cav-1DGI, d) and cav-2 (cav-2DKV, e) and the cells labeled with mouse anti-HA tag. Both equivalent mutants accumulated in morphologically identical rings to cav-3DGV, demonstrating that the CDV compartment is a general characteristic of all the caveolin family members. f, BHK cells were transfected with HA-tagged cav-3LLS (a cav-3 truncation lacking the entire NH2-terminal domain up to the putative intramembrane region) and labeled with mouse anti-HA tag. Cav-3LLS accumulated, as cav-3DGV, in CDV-like structures, showing that the caveolin scaffolding domain is not involved in the targeting of the protein to this compartment. Bars, 5 μm.
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Figure 3: The cavDGV phenotype is a feature of all caveolin family members. a–c, BHK cells were cotransfected with full-length VSV-G–tagged cav-1 and HA-tagged cavDGV, and after 24 h were labeled with a rabbit antibody to VSV-G tag and a mouse antibody to HA tag. In a low but significant proportion of transfected cells (2–5% of transfectants) full-length cav-1 (green) accumulated in the same intracellular rings as cavDGV (red). Although in those cells, cav-1 was detected on the PM (a and c, arrowheads) cavDGV was totally excluded from the PM. Some ring-like structures were strongly labeled for full-length cav-1 but not for cavDGV (b and c arrows). d and e, BHK cells were transfected with HA-tagged cav-3DGV equivalent truncation mutants of cav-1 (cav-1DGI, d) and cav-2 (cav-2DKV, e) and the cells labeled with mouse anti-HA tag. Both equivalent mutants accumulated in morphologically identical rings to cav-3DGV, demonstrating that the CDV compartment is a general characteristic of all the caveolin family members. f, BHK cells were transfected with HA-tagged cav-3LLS (a cav-3 truncation lacking the entire NH2-terminal domain up to the putative intramembrane region) and labeled with mouse anti-HA tag. Cav-3LLS accumulated, as cav-3DGV, in CDV-like structures, showing that the caveolin scaffolding domain is not involved in the targeting of the protein to this compartment. Bars, 5 μm.

Mentions: We next investigated whether the CDVs might be a general feature of the caveolin cycling pathway by examining whether full-length caveolins can access this compartment. CavDGV was coexpressed with full-length cav-1, 2, or 3. Full-length caveolins distributed in a similar manner to endogenous cav-1 and the protein could be detected on the PM, in the Golgi region of the cells, and showed limited colocalization with the cavDGV mutant protein (not shown). Extensive colocalization was observed in only a low but significant percentage of the cavDGV mutant expressing cells (2–5%, see Fig. 3 a). Full-length caveolins can reach both the PM and the CDVs but cavDGV is totally excluded from the PM (see arrowheads in Fig. 3b and Fig. c). Notice that not all the cav-1 positive rings contained cavDGV (compare Fig. 3b and Fig. c, arrows), suggesting that the formation of CDV is not exclusively induced by the presence of the mutant protein (see also Fig. 1 f). Since cav-1 does not oligomerize with cav-3, these results suggest that CDVs may be accessed by wild-type caveolins under physiological conditions.


A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance.

Pol A, Luetterforst R, Lindsay M, Heino S, Ikonen E, Parton RG - J. Cell Biol. (2001)

The cavDGV phenotype is a feature of all caveolin family members. a–c, BHK cells were cotransfected with full-length VSV-G–tagged cav-1 and HA-tagged cavDGV, and after 24 h were labeled with a rabbit antibody to VSV-G tag and a mouse antibody to HA tag. In a low but significant proportion of transfected cells (2–5% of transfectants) full-length cav-1 (green) accumulated in the same intracellular rings as cavDGV (red). Although in those cells, cav-1 was detected on the PM (a and c, arrowheads) cavDGV was totally excluded from the PM. Some ring-like structures were strongly labeled for full-length cav-1 but not for cavDGV (b and c arrows). d and e, BHK cells were transfected with HA-tagged cav-3DGV equivalent truncation mutants of cav-1 (cav-1DGI, d) and cav-2 (cav-2DKV, e) and the cells labeled with mouse anti-HA tag. Both equivalent mutants accumulated in morphologically identical rings to cav-3DGV, demonstrating that the CDV compartment is a general characteristic of all the caveolin family members. f, BHK cells were transfected with HA-tagged cav-3LLS (a cav-3 truncation lacking the entire NH2-terminal domain up to the putative intramembrane region) and labeled with mouse anti-HA tag. Cav-3LLS accumulated, as cav-3DGV, in CDV-like structures, showing that the caveolin scaffolding domain is not involved in the targeting of the protein to this compartment. Bars, 5 μm.
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Related In: Results  -  Collection

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Figure 3: The cavDGV phenotype is a feature of all caveolin family members. a–c, BHK cells were cotransfected with full-length VSV-G–tagged cav-1 and HA-tagged cavDGV, and after 24 h were labeled with a rabbit antibody to VSV-G tag and a mouse antibody to HA tag. In a low but significant proportion of transfected cells (2–5% of transfectants) full-length cav-1 (green) accumulated in the same intracellular rings as cavDGV (red). Although in those cells, cav-1 was detected on the PM (a and c, arrowheads) cavDGV was totally excluded from the PM. Some ring-like structures were strongly labeled for full-length cav-1 but not for cavDGV (b and c arrows). d and e, BHK cells were transfected with HA-tagged cav-3DGV equivalent truncation mutants of cav-1 (cav-1DGI, d) and cav-2 (cav-2DKV, e) and the cells labeled with mouse anti-HA tag. Both equivalent mutants accumulated in morphologically identical rings to cav-3DGV, demonstrating that the CDV compartment is a general characteristic of all the caveolin family members. f, BHK cells were transfected with HA-tagged cav-3LLS (a cav-3 truncation lacking the entire NH2-terminal domain up to the putative intramembrane region) and labeled with mouse anti-HA tag. Cav-3LLS accumulated, as cav-3DGV, in CDV-like structures, showing that the caveolin scaffolding domain is not involved in the targeting of the protein to this compartment. Bars, 5 μm.
Mentions: We next investigated whether the CDVs might be a general feature of the caveolin cycling pathway by examining whether full-length caveolins can access this compartment. CavDGV was coexpressed with full-length cav-1, 2, or 3. Full-length caveolins distributed in a similar manner to endogenous cav-1 and the protein could be detected on the PM, in the Golgi region of the cells, and showed limited colocalization with the cavDGV mutant protein (not shown). Extensive colocalization was observed in only a low but significant percentage of the cavDGV mutant expressing cells (2–5%, see Fig. 3 a). Full-length caveolins can reach both the PM and the CDVs but cavDGV is totally excluded from the PM (see arrowheads in Fig. 3b and Fig. c). Notice that not all the cav-1 positive rings contained cavDGV (compare Fig. 3b and Fig. c, arrows), suggesting that the formation of CDV is not exclusively induced by the presence of the mutant protein (see also Fig. 1 f). Since cav-1 does not oligomerize with cav-3, these results suggest that CDVs may be accessed by wild-type caveolins under physiological conditions.

Bottom Line: The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis.Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets.We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, Centre for Microscopy and Microanalysis and Department of Physiology and Pharmacology, University of Queensland, Queensland 4072, Australia.

ABSTRACT
Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV), specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

Show MeSH