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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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DRM isolation after CO treatment of WT and Cav1−/− MEFs. MEF cell homogenates prelabeled with [14C]acetate were left untreated or incubated with 0.5 U/ml CO for 60 min at 37°C. DRM purification was performed, and 14C-cholesterol and 14C-cholestenone were determined for each fraction. A: CO activity in total cell homogenates from treated WT and Cav1−/− MEFs. CO activity is expressed as a percentage of total [14C]cholesterol. B: Distribution of CO-insensitive [14C]cholesterol on DRM gradients for control (cells lefts untreated) and CO-treated cells for WT (top) and Cav1−/− (bottom) MEFs, respectively. Asterisk indicates a statistical significant difference of P< 0.05 relative to control. C: Distribution of CO-sensitive [14C]cholestenone on DRM gradients for WT and Cav1−/− MEFs. D: Twenty microliters of each fraction from WT and Cav1−/− MEFs treated with CO was analyzed by immunoblotting for YES, Cav1, and Cav2. The data shown in A–C are mean ± SD of three independent experiments.
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fig5: DRM isolation after CO treatment of WT and Cav1−/− MEFs. MEF cell homogenates prelabeled with [14C]acetate were left untreated or incubated with 0.5 U/ml CO for 60 min at 37°C. DRM purification was performed, and 14C-cholesterol and 14C-cholestenone were determined for each fraction. A: CO activity in total cell homogenates from treated WT and Cav1−/− MEFs. CO activity is expressed as a percentage of total [14C]cholesterol. B: Distribution of CO-insensitive [14C]cholesterol on DRM gradients for control (cells lefts untreated) and CO-treated cells for WT (top) and Cav1−/− (bottom) MEFs, respectively. Asterisk indicates a statistical significant difference of P< 0.05 relative to control. C: Distribution of CO-sensitive [14C]cholestenone on DRM gradients for WT and Cav1−/− MEFs. D: Twenty microliters of each fraction from WT and Cav1−/− MEFs treated with CO was analyzed by immunoblotting for YES, Cav1, and Cav2. The data shown in A–C are mean ± SD of three independent experiments.

Mentions: The cell-impermeable enzyme, CO, converts cholesterol to cholestenone and is widely used to probe changes in plasma membrane cholesterol distribution. There are several technical variations in the method that can lead to quantitative differences in the estimated size of the cholesterol-oxidase sensitive pool (42, 43). Previously, it was reported that a particular protocol, which avoids fixation of the cells, preferentially oxidizes cholesterol present in caveolae, causing Cav1 to relocate in the Golgi (25). This property has been used to study the role of caveolae in signal transduction processes and cholesterol transport, suggesting that Cav1 transports newly synthesized cholesterol to the plasma membrane (44). We used the same conditions to measure the CO-sensitive cholesterol pools in WT and Cav1−/− MEFs (Fig. 5). Consistent with previous studies, ∼10% of cellular cholesterol was converted to cholestenone in WT cells (Fig. 5A). Furthermore, we observed no significant difference in CO-sensitive cholesterol between WT and Cav1−/− MEFs (P > 0.05). Because Cav1−/− MEFs lack caveolae, this result suggests that CO accessible cholesterol is not restricted to, or even predominantly in, caveolae. To further explore the location of CO-sensitive cholesterol, DRMs were isolated from CO-treated WT and Cav1−/− cells. In each fraction, we measured CO-sensitive ([14C]cholestenone; Fig. 5C) and CO-insensitive ([14C]cholesterol; Fig. 5B) cholesterol. CO treatment significantly lowered the proportion of labeled cholesterol recovered in DRM (Fraction 1, Fig. 5B) in both WT and Cav1−/− MEFs. The majority of [14C]cholestenone accumulated in the non-DRM fractions of WT and Cav1−/− MEFs (Fig. 5C), and we observed no differences in cholestenone accumulation across the gradient between WT and Cav1−/− MEFs. Taken together, the data suggest that caveolae do not represent a specific or major site of action for CO.


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)

DRM isolation after CO treatment of WT and Cav1−/− MEFs. MEF cell homogenates prelabeled with [14C]acetate were left untreated or incubated with 0.5 U/ml CO for 60 min at 37°C. DRM purification was performed, and 14C-cholesterol and 14C-cholestenone were determined for each fraction. A: CO activity in total cell homogenates from treated WT and Cav1−/− MEFs. CO activity is expressed as a percentage of total [14C]cholesterol. B: Distribution of CO-insensitive [14C]cholesterol on DRM gradients for control (cells lefts untreated) and CO-treated cells for WT (top) and Cav1−/− (bottom) MEFs, respectively. Asterisk indicates a statistical significant difference of P< 0.05 relative to control. C: Distribution of CO-sensitive [14C]cholestenone on DRM gradients for WT and Cav1−/− MEFs. D: Twenty microliters of each fraction from WT and Cav1−/− MEFs treated with CO was analyzed by immunoblotting for YES, Cav1, and Cav2. The data shown in A–C are mean ± SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: DRM isolation after CO treatment of WT and Cav1−/− MEFs. MEF cell homogenates prelabeled with [14C]acetate were left untreated or incubated with 0.5 U/ml CO for 60 min at 37°C. DRM purification was performed, and 14C-cholesterol and 14C-cholestenone were determined for each fraction. A: CO activity in total cell homogenates from treated WT and Cav1−/− MEFs. CO activity is expressed as a percentage of total [14C]cholesterol. B: Distribution of CO-insensitive [14C]cholesterol on DRM gradients for control (cells lefts untreated) and CO-treated cells for WT (top) and Cav1−/− (bottom) MEFs, respectively. Asterisk indicates a statistical significant difference of P< 0.05 relative to control. C: Distribution of CO-sensitive [14C]cholestenone on DRM gradients for WT and Cav1−/− MEFs. D: Twenty microliters of each fraction from WT and Cav1−/− MEFs treated with CO was analyzed by immunoblotting for YES, Cav1, and Cav2. The data shown in A–C are mean ± SD of three independent experiments.
Mentions: The cell-impermeable enzyme, CO, converts cholesterol to cholestenone and is widely used to probe changes in plasma membrane cholesterol distribution. There are several technical variations in the method that can lead to quantitative differences in the estimated size of the cholesterol-oxidase sensitive pool (42, 43). Previously, it was reported that a particular protocol, which avoids fixation of the cells, preferentially oxidizes cholesterol present in caveolae, causing Cav1 to relocate in the Golgi (25). This property has been used to study the role of caveolae in signal transduction processes and cholesterol transport, suggesting that Cav1 transports newly synthesized cholesterol to the plasma membrane (44). We used the same conditions to measure the CO-sensitive cholesterol pools in WT and Cav1−/− MEFs (Fig. 5). Consistent with previous studies, ∼10% of cellular cholesterol was converted to cholestenone in WT cells (Fig. 5A). Furthermore, we observed no significant difference in CO-sensitive cholesterol between WT and Cav1−/− MEFs (P > 0.05). Because Cav1−/− MEFs lack caveolae, this result suggests that CO accessible cholesterol is not restricted to, or even predominantly in, caveolae. To further explore the location of CO-sensitive cholesterol, DRMs were isolated from CO-treated WT and Cav1−/− cells. In each fraction, we measured CO-sensitive ([14C]cholestenone; Fig. 5C) and CO-insensitive ([14C]cholesterol; Fig. 5B) cholesterol. CO treatment significantly lowered the proportion of labeled cholesterol recovered in DRM (Fraction 1, Fig. 5B) in both WT and Cav1−/− MEFs. The majority of [14C]cholestenone accumulated in the non-DRM fractions of WT and Cav1−/− MEFs (Fig. 5C), and we observed no differences in cholestenone accumulation across the gradient between WT and Cav1−/− MEFs. Taken together, the data suggest that caveolae do not represent a specific or major site of action for CO.

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
Related in: MedlinePlus