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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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Ordered and fluid domains in WT and Cav1−/− MEFs. A: WT and Cav1−/− MEFs were labeled with the fluorescent membrane dye Laurdan. Laurdan intensity was converted into the fluidity index GP. GP values range from −1 (most fluid) to + 1 (most ordered). From the GP images, the GP distribution (open diamond, shown for WT MEFs) is obtained, which is fitted to two Gaussian populations (line through data). Black vertical lines denote the centers of the fluid population (Pf), and gray vertical lines denote the centers of the ordered populations (Po). Center values and coverages (area under the curve for each population) are given for both populations. B, C: The mean GP value of fluid (B) and ordered (C) membranes and their relative abundance (percentage of coverage) are shown. WT MEFs (diamond), Cav1−/− MEFs (square), Cav1−/− MEFs transfected with WT Cav1 (triangle), and Cav1−/− MEFs expressing mutant Y14FCav1 (circle) were analyzed. Error bars are SDs of three independent experiments with ∼25 cells per experiments.
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fig6: Ordered and fluid domains in WT and Cav1−/− MEFs. A: WT and Cav1−/− MEFs were labeled with the fluorescent membrane dye Laurdan. Laurdan intensity was converted into the fluidity index GP. GP values range from −1 (most fluid) to + 1 (most ordered). From the GP images, the GP distribution (open diamond, shown for WT MEFs) is obtained, which is fitted to two Gaussian populations (line through data). Black vertical lines denote the centers of the fluid population (Pf), and gray vertical lines denote the centers of the ordered populations (Po). Center values and coverages (area under the curve for each population) are given for both populations. B, C: The mean GP value of fluid (B) and ordered (C) membranes and their relative abundance (percentage of coverage) are shown. WT MEFs (diamond), Cav1−/− MEFs (square), Cav1−/− MEFs transfected with WT Cav1 (triangle), and Cav1−/− MEFs expressing mutant Y14FCav1 (circle) were analyzed. Error bars are SDs of three independent experiments with ∼25 cells per experiments.

Mentions: For a more general analysis of the effects of Cav1 on membrane structure, we compared the global GP distribution in MEFs, shown in Fig. 6A for WT MEFs. Two populations, fluid (Pf, dark gray) and ordered (Po, light gray), were identified. Each population has a characteristic mean GP value and abundance (or coverage) that equate to the area under the curve (34). The mean GP values of fluid membranes, Pf, were similar in WT and Cav1−/− MEFs (Fig. 6B), but in Cav1−/− cells covered a greater proportion of the membrane surface (91.9 ± 3.7% in Cav1−/− MEFs vs. 71.4 ± 5.7% in WT MEFs; y axis in Fig. 6B). In contrast, the ordered membranes were relatively more fluid and less abundant in Cav1−/− MEFs than WT MEFs (Fig. 6C), suggesting that Cav1 does contribute to the lipid order of raft-like domains in adherent cells. To demonstrate that the differences in membrane structure were directly related to Cav1 expression and function, Cav1−/− MEFs were transfected with either WT Cav1 or with Y14F Cav1. Expressing WT Cav1 in Cav1−/− MEFs restored both order and relative surface coverage of ordered domains to WT levels. Mutating the only tyrosine phosphorylation site in Cav1 (Y14F Cav1) has no effect on caveolae formation but inhibits raft internalization (46) and decreases membrane order at focal adhesions (34). Expression of Y14F Cav1 only partially rescued ordered domains when expressed in Cav1−/− MEF (Fig. 6C). WT Cav1 and Y14F Cav1 correspondingly decreased the proportion of fluid domains in Cav1−/− MEF (Fig. 6B). Overall, the data support a contribution of Cav1 to the generation of ordered domains in adherent, intact cells. The differential effect of Cav1 expression on biochemically identified domains and those observed by microscopy is an indication that cell architecture plays a greater role in lipid raft abundance than subtle changes in lipid composition.


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)

Ordered and fluid domains in WT and Cav1−/− MEFs. A: WT and Cav1−/− MEFs were labeled with the fluorescent membrane dye Laurdan. Laurdan intensity was converted into the fluidity index GP. GP values range from −1 (most fluid) to + 1 (most ordered). From the GP images, the GP distribution (open diamond, shown for WT MEFs) is obtained, which is fitted to two Gaussian populations (line through data). Black vertical lines denote the centers of the fluid population (Pf), and gray vertical lines denote the centers of the ordered populations (Po). Center values and coverages (area under the curve for each population) are given for both populations. B, C: The mean GP value of fluid (B) and ordered (C) membranes and their relative abundance (percentage of coverage) are shown. WT MEFs (diamond), Cav1−/− MEFs (square), Cav1−/− MEFs transfected with WT Cav1 (triangle), and Cav1−/− MEFs expressing mutant Y14FCav1 (circle) were analyzed. Error bars are SDs of three independent experiments with ∼25 cells per experiments.
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fig6: Ordered and fluid domains in WT and Cav1−/− MEFs. A: WT and Cav1−/− MEFs were labeled with the fluorescent membrane dye Laurdan. Laurdan intensity was converted into the fluidity index GP. GP values range from −1 (most fluid) to + 1 (most ordered). From the GP images, the GP distribution (open diamond, shown for WT MEFs) is obtained, which is fitted to two Gaussian populations (line through data). Black vertical lines denote the centers of the fluid population (Pf), and gray vertical lines denote the centers of the ordered populations (Po). Center values and coverages (area under the curve for each population) are given for both populations. B, C: The mean GP value of fluid (B) and ordered (C) membranes and their relative abundance (percentage of coverage) are shown. WT MEFs (diamond), Cav1−/− MEFs (square), Cav1−/− MEFs transfected with WT Cav1 (triangle), and Cav1−/− MEFs expressing mutant Y14FCav1 (circle) were analyzed. Error bars are SDs of three independent experiments with ∼25 cells per experiments.
Mentions: For a more general analysis of the effects of Cav1 on membrane structure, we compared the global GP distribution in MEFs, shown in Fig. 6A for WT MEFs. Two populations, fluid (Pf, dark gray) and ordered (Po, light gray), were identified. Each population has a characteristic mean GP value and abundance (or coverage) that equate to the area under the curve (34). The mean GP values of fluid membranes, Pf, were similar in WT and Cav1−/− MEFs (Fig. 6B), but in Cav1−/− cells covered a greater proportion of the membrane surface (91.9 ± 3.7% in Cav1−/− MEFs vs. 71.4 ± 5.7% in WT MEFs; y axis in Fig. 6B). In contrast, the ordered membranes were relatively more fluid and less abundant in Cav1−/− MEFs than WT MEFs (Fig. 6C), suggesting that Cav1 does contribute to the lipid order of raft-like domains in adherent cells. To demonstrate that the differences in membrane structure were directly related to Cav1 expression and function, Cav1−/− MEFs were transfected with either WT Cav1 or with Y14F Cav1. Expressing WT Cav1 in Cav1−/− MEFs restored both order and relative surface coverage of ordered domains to WT levels. Mutating the only tyrosine phosphorylation site in Cav1 (Y14F Cav1) has no effect on caveolae formation but inhibits raft internalization (46) and decreases membrane order at focal adhesions (34). Expression of Y14F Cav1 only partially rescued ordered domains when expressed in Cav1−/− MEF (Fig. 6C). WT Cav1 and Y14F Cav1 correspondingly decreased the proportion of fluid domains in Cav1−/− MEF (Fig. 6B). Overall, the data support a contribution of Cav1 to the generation of ordered domains in adherent, intact cells. The differential effect of Cav1 expression on biochemically identified domains and those observed by microscopy is an indication that cell architecture plays a greater role in lipid raft abundance than subtle changes in lipid composition.

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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