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Caveolin-1-dependent and -independent membrane domains.

Le Lay S, Li Q, Proschogo N, Rodriguez M, Gunaratnam K, Cartland S, Rentero C, Jessup W, Mitchell T, Gaus K - J. Lipid Res. (2008)

Bottom Line: Our findings show that Cav1 expression had no effect on free (membrane-associated) cholesterol levels.Despite differences in phospholipid composition, we found that cholesterol levels in DRMs, NDR, and CO-sensitive domains were similar in both cell types.The data suggest that Cav1 is not required to target cholesterol to lipid rafts and that CO does not specifically oxidize caveolar cholesterol.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche des Cordeliers, INSERM, U872, Université Pierre et Marie Curie, Paris 6, France.

ABSTRACT
Lipid rafts defined as cholesterol- and sphingomyelin-rich domains have been isolated from different cell types that vary greatly in their lipid profiles. Here, we investigated the contribution of the structural protein caveolin-1 (Cav1) to the overall lipid composition and domain abundance in mouse embryonic fibroblasts (MEFs) from wild-type (WT) or Cav1-deficient (Cav1(-/-)) animals. Our findings show that Cav1 expression had no effect on free (membrane-associated) cholesterol levels. However, Cav1(-/-)-deficient cells did have a higher proportion of sphingomyelin, decreased abundance of unsaturated phospholipids, and a trend toward shorter fatty acid chains in phosphatidylcholine. We isolated detergent-resistant membranes (DRMs), nondetergent raft domains (NDR), and cholesterol oxidase (CO)-sensitive domains and assessed the abundance of ordered domains in intact cells using the fluorescent dye Laurdan. Despite differences in phospholipid composition, we found that cholesterol levels in DRMs, NDR, and CO-sensitive domains were similar in both cell types. The data suggest that Cav1 is not required to target cholesterol to lipid rafts and that CO does not specifically oxidize caveolar cholesterol. In contrast, the abundance of ordered domains in adherent cells is reduced in Cav1(-/-) compared with WT MEFs, suggesting that cell architecture is critical in maintaining Cav1-induced lipid rafts.

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Cholesterol and PL content of WT and Cav1−/− MEFs. A, B: Unesterified (free; A) and esterified cholesterol (B) was analyzed by HPLC and normalized to cell protein. Values are mean and standard deviation of six samples each. Asterisk in B indicates a significant difference of P< 0.05 between WT and Cav1−/− MEFs. C: Relative abundance of PLs in WT (closed bars) and Cav1−/− (open bars) MEFs. Cells were homogenates, lipids extracted and analyzed by mass spectrometry as described in Materials and Methods. Total PL mass was 333 ± 27 nmol/mg cell protein and 199 ± 36 nmol/mg in WT and Cav1−/− MEFs, respectively. D: Relative levels of PC esters in WT (closed bars) and Cav1−/− (open bars) MEFs. E: PL synthesis in WT (closed bars) and Cav1−/− (open bars) MEFs is measured by incorporation of 14C-choline over a 24 h period. Radioactivity (CPM) is normalized to cell protein. A–E: Asterisks indicate a significant difference between WT and Cav1−/− MEFs of P< 0.05. Data are presented as mean + SE (n = 4).
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fig2: Cholesterol and PL content of WT and Cav1−/− MEFs. A, B: Unesterified (free; A) and esterified cholesterol (B) was analyzed by HPLC and normalized to cell protein. Values are mean and standard deviation of six samples each. Asterisk in B indicates a significant difference of P< 0.05 between WT and Cav1−/− MEFs. C: Relative abundance of PLs in WT (closed bars) and Cav1−/− (open bars) MEFs. Cells were homogenates, lipids extracted and analyzed by mass spectrometry as described in Materials and Methods. Total PL mass was 333 ± 27 nmol/mg cell protein and 199 ± 36 nmol/mg in WT and Cav1−/− MEFs, respectively. D: Relative levels of PC esters in WT (closed bars) and Cav1−/− (open bars) MEFs. E: PL synthesis in WT (closed bars) and Cav1−/− (open bars) MEFs is measured by incorporation of 14C-choline over a 24 h period. Radioactivity (CPM) is normalized to cell protein. A–E: Asterisks indicate a significant difference between WT and Cav1−/− MEFs of P< 0.05. Data are presented as mean + SE (n = 4).

Mentions: We found no significant difference in total and free cholesterol levels between Cav1−/− and WT MEF (Fig. 2A; P > 0.05). This suggests that membrane cholesterol levels are similar in both cell types despite a significant reduction in cholesterol synthesis in Cav1−/− MEF (data not shown). In Cav1−/− MEF, a significantly higher proportion of cholesterol is present as cholesterol esters compared with WT MEFs (Fig. 2B; P < 0.05). In summary, Cav1 expression in MEFs alters the balance between free and esterified cholesterol, although the total cholesterol levels were not significantly different. A previous report suggested that Cav1 plays a role in regulating intracellular cholesterol because free cholesterol levels decreased and esterified cholesterol increased in Cav1−/− compared with WT MEFs (39).


Caveolin-1-dependent and -independent membrane domains.

Le Lay S, Li Q, Proschogo N, Rodriguez M, Gunaratnam K, Cartland S, Rentero C, Jessup W, Mitchell T, Gaus K - J. Lipid Res. (2008)

Cholesterol and PL content of WT and Cav1−/− MEFs. A, B: Unesterified (free; A) and esterified cholesterol (B) was analyzed by HPLC and normalized to cell protein. Values are mean and standard deviation of six samples each. Asterisk in B indicates a significant difference of P< 0.05 between WT and Cav1−/− MEFs. C: Relative abundance of PLs in WT (closed bars) and Cav1−/− (open bars) MEFs. Cells were homogenates, lipids extracted and analyzed by mass spectrometry as described in Materials and Methods. Total PL mass was 333 ± 27 nmol/mg cell protein and 199 ± 36 nmol/mg in WT and Cav1−/− MEFs, respectively. D: Relative levels of PC esters in WT (closed bars) and Cav1−/− (open bars) MEFs. E: PL synthesis in WT (closed bars) and Cav1−/− (open bars) MEFs is measured by incorporation of 14C-choline over a 24 h period. Radioactivity (CPM) is normalized to cell protein. A–E: Asterisks indicate a significant difference between WT and Cav1−/− MEFs of P< 0.05. Data are presented as mean + SE (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724060&req=5

fig2: Cholesterol and PL content of WT and Cav1−/− MEFs. A, B: Unesterified (free; A) and esterified cholesterol (B) was analyzed by HPLC and normalized to cell protein. Values are mean and standard deviation of six samples each. Asterisk in B indicates a significant difference of P< 0.05 between WT and Cav1−/− MEFs. C: Relative abundance of PLs in WT (closed bars) and Cav1−/− (open bars) MEFs. Cells were homogenates, lipids extracted and analyzed by mass spectrometry as described in Materials and Methods. Total PL mass was 333 ± 27 nmol/mg cell protein and 199 ± 36 nmol/mg in WT and Cav1−/− MEFs, respectively. D: Relative levels of PC esters in WT (closed bars) and Cav1−/− (open bars) MEFs. E: PL synthesis in WT (closed bars) and Cav1−/− (open bars) MEFs is measured by incorporation of 14C-choline over a 24 h period. Radioactivity (CPM) is normalized to cell protein. A–E: Asterisks indicate a significant difference between WT and Cav1−/− MEFs of P< 0.05. Data are presented as mean + SE (n = 4).
Mentions: We found no significant difference in total and free cholesterol levels between Cav1−/− and WT MEF (Fig. 2A; P > 0.05). This suggests that membrane cholesterol levels are similar in both cell types despite a significant reduction in cholesterol synthesis in Cav1−/− MEF (data not shown). In Cav1−/− MEF, a significantly higher proportion of cholesterol is present as cholesterol esters compared with WT MEFs (Fig. 2B; P < 0.05). In summary, Cav1 expression in MEFs alters the balance between free and esterified cholesterol, although the total cholesterol levels were not significantly different. A previous report suggested that Cav1 plays a role in regulating intracellular cholesterol because free cholesterol levels decreased and esterified cholesterol increased in Cav1−/− compared with WT MEFs (39).

Bottom Line: Our findings show that Cav1 expression had no effect on free (membrane-associated) cholesterol levels.Despite differences in phospholipid composition, we found that cholesterol levels in DRMs, NDR, and CO-sensitive domains were similar in both cell types.The data suggest that Cav1 is not required to target cholesterol to lipid rafts and that CO does not specifically oxidize caveolar cholesterol.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche des Cordeliers, INSERM, U872, Université Pierre et Marie Curie, Paris 6, France.

ABSTRACT
Lipid rafts defined as cholesterol- and sphingomyelin-rich domains have been isolated from different cell types that vary greatly in their lipid profiles. Here, we investigated the contribution of the structural protein caveolin-1 (Cav1) to the overall lipid composition and domain abundance in mouse embryonic fibroblasts (MEFs) from wild-type (WT) or Cav1-deficient (Cav1(-/-)) animals. Our findings show that Cav1 expression had no effect on free (membrane-associated) cholesterol levels. However, Cav1(-/-)-deficient cells did have a higher proportion of sphingomyelin, decreased abundance of unsaturated phospholipids, and a trend toward shorter fatty acid chains in phosphatidylcholine. We isolated detergent-resistant membranes (DRMs), nondetergent raft domains (NDR), and cholesterol oxidase (CO)-sensitive domains and assessed the abundance of ordered domains in intact cells using the fluorescent dye Laurdan. Despite differences in phospholipid composition, we found that cholesterol levels in DRMs, NDR, and CO-sensitive domains were similar in both cell types. The data suggest that Cav1 is not required to target cholesterol to lipid rafts and that CO does not specifically oxidize caveolar cholesterol. In contrast, the abundance of ordered domains in adherent cells is reduced in Cav1(-/-) compared with WT MEFs, suggesting that cell architecture is critical in maintaining Cav1-induced lipid rafts.

Show MeSH