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Reversible Dissolution of Microdomains in Detergent-Resistant Membranes at Physiological Temperature.

Cremona A, Orsini F, Corsetto PA, Hoogenboom BW, Rizzo AM - PLoS ONE (2015)

Bottom Line: This shrinking in microdomain size was accompanied by a gradual reduction of the height difference between the microdomains and the surrounding membrane, consistent with the behaviour expected for lipids that are laterally segregated in liquid ordered and liquid disordered domains.Immunolabeling experiments demonstrated that the microdomains contained flotillin-1, a protein associated with lipid rafts.The microdomains reversibly dissolved and reappeared, respectively, on heating to and cooling below temperatures around 37 °C, which is indicative of radical changes in local membrane order close to physiological temperature.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milano, Italy.

ABSTRACT
The formation of lipid microdomains ("rafts") is presumed to play an important role in various cellular functions, but their nature remains controversial. Here we report on microdomain formation in isolated, detergent-resistant membranes from MDA-MB-231 human breast cancer cells, studied by atomic force microscopy (AFM). Whereas microdomains were readily observed at room temperature, they shrunk in size and mostly disappeared at higher temperatures. This shrinking in microdomain size was accompanied by a gradual reduction of the height difference between the microdomains and the surrounding membrane, consistent with the behaviour expected for lipids that are laterally segregated in liquid ordered and liquid disordered domains. Immunolabeling experiments demonstrated that the microdomains contained flotillin-1, a protein associated with lipid rafts. The microdomains reversibly dissolved and reappeared, respectively, on heating to and cooling below temperatures around 37 °C, which is indicative of radical changes in local membrane order close to physiological temperature.

No MeSH data available.


Related in: MedlinePlus

Microdomains in detergent-resistant membranes.a, AFM topography of detergent-resistant membranes (isolated on sucrose gradient, fraction 5) adsorbed on a mica substrate (dark brown), recorded in buffer solution at 25°C. Microdomains appear as elevated (brighter) plateaus with lateral dimensions of 100–300 nm on top of the membrane patches. b, Height profile corresponding to the white line drawn in (a).
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pone.0132696.g001: Microdomains in detergent-resistant membranes.a, AFM topography of detergent-resistant membranes (isolated on sucrose gradient, fraction 5) adsorbed on a mica substrate (dark brown), recorded in buffer solution at 25°C. Microdomains appear as elevated (brighter) plateaus with lateral dimensions of 100–300 nm on top of the membrane patches. b, Height profile corresponding to the white line drawn in (a).

Mentions: Detergent-resistant membranes were purified from MDA-MB-231 human breast cancer cells by ultracentrifugation on a discontinuous sucrose gradient. For low-density fractions (fractions 5 and 6) at the 5% and 30% sucrose interface, the isolated membranes were enriched in cholesterol and sphingomyelin, as demonstrated by High-Performance Thin Layer Chromatography, and Western Blotting of these membranes (S1 Fig) demonstrated the presence of flotillin-1, a protein associated with lipid rafts [31]. The results presented here all refer to membranes from fraction 5. Imaged by AFM in aqueous solution and at room temperature, the isolated membrane samples appeared as patches of 4 nm height–the approximate thickness of a lipid bilayer–adsorbed a mica substrate (Fig 1). As reported previously [31], these membrane patches contained static microdomains with lateral dimensions in the range of 100–300 nm, protruding 1–2 nm from the patch surface, and with increased roughness compared to the surrounding membrane.


Reversible Dissolution of Microdomains in Detergent-Resistant Membranes at Physiological Temperature.

Cremona A, Orsini F, Corsetto PA, Hoogenboom BW, Rizzo AM - PLoS ONE (2015)

Microdomains in detergent-resistant membranes.a, AFM topography of detergent-resistant membranes (isolated on sucrose gradient, fraction 5) adsorbed on a mica substrate (dark brown), recorded in buffer solution at 25°C. Microdomains appear as elevated (brighter) plateaus with lateral dimensions of 100–300 nm on top of the membrane patches. b, Height profile corresponding to the white line drawn in (a).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493071&req=5

pone.0132696.g001: Microdomains in detergent-resistant membranes.a, AFM topography of detergent-resistant membranes (isolated on sucrose gradient, fraction 5) adsorbed on a mica substrate (dark brown), recorded in buffer solution at 25°C. Microdomains appear as elevated (brighter) plateaus with lateral dimensions of 100–300 nm on top of the membrane patches. b, Height profile corresponding to the white line drawn in (a).
Mentions: Detergent-resistant membranes were purified from MDA-MB-231 human breast cancer cells by ultracentrifugation on a discontinuous sucrose gradient. For low-density fractions (fractions 5 and 6) at the 5% and 30% sucrose interface, the isolated membranes were enriched in cholesterol and sphingomyelin, as demonstrated by High-Performance Thin Layer Chromatography, and Western Blotting of these membranes (S1 Fig) demonstrated the presence of flotillin-1, a protein associated with lipid rafts [31]. The results presented here all refer to membranes from fraction 5. Imaged by AFM in aqueous solution and at room temperature, the isolated membrane samples appeared as patches of 4 nm height–the approximate thickness of a lipid bilayer–adsorbed a mica substrate (Fig 1). As reported previously [31], these membrane patches contained static microdomains with lateral dimensions in the range of 100–300 nm, protruding 1–2 nm from the patch surface, and with increased roughness compared to the surrounding membrane.

Bottom Line: This shrinking in microdomain size was accompanied by a gradual reduction of the height difference between the microdomains and the surrounding membrane, consistent with the behaviour expected for lipids that are laterally segregated in liquid ordered and liquid disordered domains.Immunolabeling experiments demonstrated that the microdomains contained flotillin-1, a protein associated with lipid rafts.The microdomains reversibly dissolved and reappeared, respectively, on heating to and cooling below temperatures around 37 °C, which is indicative of radical changes in local membrane order close to physiological temperature.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milano, Italy.

ABSTRACT
The formation of lipid microdomains ("rafts") is presumed to play an important role in various cellular functions, but their nature remains controversial. Here we report on microdomain formation in isolated, detergent-resistant membranes from MDA-MB-231 human breast cancer cells, studied by atomic force microscopy (AFM). Whereas microdomains were readily observed at room temperature, they shrunk in size and mostly disappeared at higher temperatures. This shrinking in microdomain size was accompanied by a gradual reduction of the height difference between the microdomains and the surrounding membrane, consistent with the behaviour expected for lipids that are laterally segregated in liquid ordered and liquid disordered domains. Immunolabeling experiments demonstrated that the microdomains contained flotillin-1, a protein associated with lipid rafts. The microdomains reversibly dissolved and reappeared, respectively, on heating to and cooling below temperatures around 37 °C, which is indicative of radical changes in local membrane order close to physiological temperature.

No MeSH data available.


Related in: MedlinePlus