Limits...
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.

Show MeSH

Related in: MedlinePlus

CDV is a lipid-enriched compartment and cavDGV expression induces lipid accumulation. a–c, CavDGV HA-tagged transfected cells were labeled with mAbs to HA, followed by Cy3-conjugated secondary antibodies. The samples were mounted in mowiol, containing a solution of the lipid probe, Nile red. The cells were selected for the presence of rings in the red channel and the corresponding image in the green channel was captured. Then the samples were exposed for 30 s in the red channel before the corresponding image was captured. Nile red staining was within the center of CDVs, whereas cavDGV formed the characteristic ring around the vesicle (arrowheads and merge panel). Cells expressing cavDGV showed a marked accumulation of lipids, in CDVs, and in other vesicular structures (arrows in c), compared with nontransfected cells. d–f, CavDGV HA-tagged transfected cells were labeled with filipin (to visualize cellular FC, see Materials and Methods for details) and with an mAb to the HA tag. CavDGV expression induced a marked redistribution of filipin: 65% of cavDGV expressing cells did not contain filipin on the PM (arrows in d and f) and in 72% of the transfected cells filipin was detected in very enlarged cytoplasmic vesicles (arrowheads and insert f). g–j, CavDGV HA-tagged transfected cells were labeled with filipin (blue) and with antibodies to the HA tag (red) and to LBPA (green). CavDGV expression induced redistribution of FC to very enlarged late endosomes (arrows in i and j). k–m, BHK were transfected with cavDGV in a normal medium (k) or in a medium containing 4.5 μg/ml of U18666A (l and m) for different times and cholesterol distribution was monitored by means of filipin. In cells expressing cavDGV, a complete accumulation of lipids in late endosomes was evident after 24–32 h (k). However, in those cells expressing cavDGV in the presence of U18666A the complete accumulation of FC in late endosomes was evident after 16 h (l), demonstrating that the combination of the drug and the cavDGV results in an earlier lysosomal storage disorder. Finally, after 24 h few differences could be observed between nonexpressing and cavDGV expressing cells, but some very enlarged intracellular vesicles were present in transfected cells (arrows in m). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198820&req=5

Figure 6: CDV is a lipid-enriched compartment and cavDGV expression induces lipid accumulation. a–c, CavDGV HA-tagged transfected cells were labeled with mAbs to HA, followed by Cy3-conjugated secondary antibodies. The samples were mounted in mowiol, containing a solution of the lipid probe, Nile red. The cells were selected for the presence of rings in the red channel and the corresponding image in the green channel was captured. Then the samples were exposed for 30 s in the red channel before the corresponding image was captured. Nile red staining was within the center of CDVs, whereas cavDGV formed the characteristic ring around the vesicle (arrowheads and merge panel). Cells expressing cavDGV showed a marked accumulation of lipids, in CDVs, and in other vesicular structures (arrows in c), compared with nontransfected cells. d–f, CavDGV HA-tagged transfected cells were labeled with filipin (to visualize cellular FC, see Materials and Methods for details) and with an mAb to the HA tag. CavDGV expression induced a marked redistribution of filipin: 65% of cavDGV expressing cells did not contain filipin on the PM (arrows in d and f) and in 72% of the transfected cells filipin was detected in very enlarged cytoplasmic vesicles (arrowheads and insert f). g–j, CavDGV HA-tagged transfected cells were labeled with filipin (blue) and with antibodies to the HA tag (red) and to LBPA (green). CavDGV expression induced redistribution of FC to very enlarged late endosomes (arrows in i and j). k–m, BHK were transfected with cavDGV in a normal medium (k) or in a medium containing 4.5 μg/ml of U18666A (l and m) for different times and cholesterol distribution was monitored by means of filipin. In cells expressing cavDGV, a complete accumulation of lipids in late endosomes was evident after 24–32 h (k). However, in those cells expressing cavDGV in the presence of U18666A the complete accumulation of FC in late endosomes was evident after 16 h (l), demonstrating that the combination of the drug and the cavDGV results in an earlier lysosomal storage disorder. Finally, after 24 h few differences could be observed between nonexpressing and cavDGV expressing cells, but some very enlarged intracellular vesicles were present in transfected cells (arrows in m). Bar, 5 μm.

Mentions: CavDGV specifically inhibits H-ras function through an effect on cholesterol at the plasma membrane. Therefore, we examined whether cavDGV had functional effects on lipid distribution. CavDGV-transfected cells were labeled with the neutral lipid probe, Nile red. When cavDGV cells were incubated with Nile red, the lipid marker accumulated in late endosomes, colocalizing with LBPA (not shown), but also in CDVs (Fig. 6 a). Close examination of the images revealed that the Nile red staining was within the center of the CDVs, whereas the caveolin mutant formed a characteristic ring around the vesicle (Fig. 6 a, insert and arrowheads) entirely consistent with the electron microscopic observations. The association of lipid droplets with a domain of the ER is consistent with recent studies in adipocytes (Prattes et al. 2000). As shown in Fig. 6 c, cavDGV expression caused an increased intracellular accumulation of lipids compared with nontransfected cells (Fig. 6b and Fig. c), demonstrating that cavDGV expression induces a lipid storage disorder by accumulating neutral lipids inside the CDVs (arrowheads) and in other intracellular compartments (arrows). Although the intracellular accumulation of lipids induced by cavDGV was variable, the mean increase in Nile red staining in cavDGV-transfected cells was over twofold higher than nontransfected cells (ratio of pixels in cavDGV-transfected/untransfected cells = 2.22 ± 0.89). In contrast, full-length cav-3 caused no significant accumulation of neutral lipids (ratio of cav-3-transfected/untransfected cells = 1.07 ± 0.10). Together with the electron microscopic observations, these results strongly suggest that the mutant caveolin protein specifically accumulates in the membrane of lipid droplets and that the mutant protein induces increased accumulation of neutral lipids.


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)

CDV is a lipid-enriched compartment and cavDGV expression induces lipid accumulation. a–c, CavDGV HA-tagged transfected cells were labeled with mAbs to HA, followed by Cy3-conjugated secondary antibodies. The samples were mounted in mowiol, containing a solution of the lipid probe, Nile red. The cells were selected for the presence of rings in the red channel and the corresponding image in the green channel was captured. Then the samples were exposed for 30 s in the red channel before the corresponding image was captured. Nile red staining was within the center of CDVs, whereas cavDGV formed the characteristic ring around the vesicle (arrowheads and merge panel). Cells expressing cavDGV showed a marked accumulation of lipids, in CDVs, and in other vesicular structures (arrows in c), compared with nontransfected cells. d–f, CavDGV HA-tagged transfected cells were labeled with filipin (to visualize cellular FC, see Materials and Methods for details) and with an mAb to the HA tag. CavDGV expression induced a marked redistribution of filipin: 65% of cavDGV expressing cells did not contain filipin on the PM (arrows in d and f) and in 72% of the transfected cells filipin was detected in very enlarged cytoplasmic vesicles (arrowheads and insert f). g–j, CavDGV HA-tagged transfected cells were labeled with filipin (blue) and with antibodies to the HA tag (red) and to LBPA (green). CavDGV expression induced redistribution of FC to very enlarged late endosomes (arrows in i and j). k–m, BHK were transfected with cavDGV in a normal medium (k) or in a medium containing 4.5 μg/ml of U18666A (l and m) for different times and cholesterol distribution was monitored by means of filipin. In cells expressing cavDGV, a complete accumulation of lipids in late endosomes was evident after 24–32 h (k). However, in those cells expressing cavDGV in the presence of U18666A the complete accumulation of FC in late endosomes was evident after 16 h (l), demonstrating that the combination of the drug and the cavDGV results in an earlier lysosomal storage disorder. Finally, after 24 h few differences could be observed between nonexpressing and cavDGV expressing cells, but some very enlarged intracellular vesicles were present in transfected cells (arrows in m). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198820&req=5

Figure 6: CDV is a lipid-enriched compartment and cavDGV expression induces lipid accumulation. a–c, CavDGV HA-tagged transfected cells were labeled with mAbs to HA, followed by Cy3-conjugated secondary antibodies. The samples were mounted in mowiol, containing a solution of the lipid probe, Nile red. The cells were selected for the presence of rings in the red channel and the corresponding image in the green channel was captured. Then the samples were exposed for 30 s in the red channel before the corresponding image was captured. Nile red staining was within the center of CDVs, whereas cavDGV formed the characteristic ring around the vesicle (arrowheads and merge panel). Cells expressing cavDGV showed a marked accumulation of lipids, in CDVs, and in other vesicular structures (arrows in c), compared with nontransfected cells. d–f, CavDGV HA-tagged transfected cells were labeled with filipin (to visualize cellular FC, see Materials and Methods for details) and with an mAb to the HA tag. CavDGV expression induced a marked redistribution of filipin: 65% of cavDGV expressing cells did not contain filipin on the PM (arrows in d and f) and in 72% of the transfected cells filipin was detected in very enlarged cytoplasmic vesicles (arrowheads and insert f). g–j, CavDGV HA-tagged transfected cells were labeled with filipin (blue) and with antibodies to the HA tag (red) and to LBPA (green). CavDGV expression induced redistribution of FC to very enlarged late endosomes (arrows in i and j). k–m, BHK were transfected with cavDGV in a normal medium (k) or in a medium containing 4.5 μg/ml of U18666A (l and m) for different times and cholesterol distribution was monitored by means of filipin. In cells expressing cavDGV, a complete accumulation of lipids in late endosomes was evident after 24–32 h (k). However, in those cells expressing cavDGV in the presence of U18666A the complete accumulation of FC in late endosomes was evident after 16 h (l), demonstrating that the combination of the drug and the cavDGV results in an earlier lysosomal storage disorder. Finally, after 24 h few differences could be observed between nonexpressing and cavDGV expressing cells, but some very enlarged intracellular vesicles were present in transfected cells (arrows in m). Bar, 5 μm.
Mentions: CavDGV specifically inhibits H-ras function through an effect on cholesterol at the plasma membrane. Therefore, we examined whether cavDGV had functional effects on lipid distribution. CavDGV-transfected cells were labeled with the neutral lipid probe, Nile red. When cavDGV cells were incubated with Nile red, the lipid marker accumulated in late endosomes, colocalizing with LBPA (not shown), but also in CDVs (Fig. 6 a). Close examination of the images revealed that the Nile red staining was within the center of the CDVs, whereas the caveolin mutant formed a characteristic ring around the vesicle (Fig. 6 a, insert and arrowheads) entirely consistent with the electron microscopic observations. The association of lipid droplets with a domain of the ER is consistent with recent studies in adipocytes (Prattes et al. 2000). As shown in Fig. 6 c, cavDGV expression caused an increased intracellular accumulation of lipids compared with nontransfected cells (Fig. 6b and Fig. c), demonstrating that cavDGV expression induces a lipid storage disorder by accumulating neutral lipids inside the CDVs (arrowheads) and in other intracellular compartments (arrows). Although the intracellular accumulation of lipids induced by cavDGV was variable, the mean increase in Nile red staining in cavDGV-transfected cells was over twofold higher than nontransfected cells (ratio of pixels in cavDGV-transfected/untransfected cells = 2.22 ± 0.89). In contrast, full-length cav-3 caused no significant accumulation of neutral lipids (ratio of cav-3-transfected/untransfected cells = 1.07 ± 0.10). Together with the electron microscopic observations, these results strongly suggest that the mutant caveolin protein specifically accumulates in the membrane of lipid droplets and that the mutant protein induces increased accumulation of neutral lipids.

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
Related in: MedlinePlus