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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

Reversible dissolution and formation of microdomains.a, AFM topography of isolated membrane samples in buffer solution, for a thermal cycle in which the temperature is first increased from 25°C to 30°C and then 37°C, followed by a decrease back to 25°C. Arrows indicate the sequence of the images. Vertical (color) scale for all AFM images: 9 nm, see also scale bar in Fig 1. b, Histogram of the surface area of the microdomains observed on the membrane patches in (a), demonstrating a shrinking of microdomains for higher temperatures and a full recovery at the end of the thermal cycle (25°C*). c, Histogram of the total surface area of all patches that are fully included within the frames displayed in (a), showing that the absolute changes in total patch area are small compared to the absolute changes in microdomain area over the thermal cycle. Error bars = 5% (see Methods).
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pone.0132696.g002: Reversible dissolution and formation of microdomains.a, AFM topography of isolated membrane samples in buffer solution, for a thermal cycle in which the temperature is first increased from 25°C to 30°C and then 37°C, followed by a decrease back to 25°C. Arrows indicate the sequence of the images. Vertical (color) scale for all AFM images: 9 nm, see also scale bar in Fig 1. b, Histogram of the surface area of the microdomains observed on the membrane patches in (a), demonstrating a shrinking of microdomains for higher temperatures and a full recovery at the end of the thermal cycle (25°C*). c, Histogram of the total surface area of all patches that are fully included within the frames displayed in (a), showing that the absolute changes in total patch area are small compared to the absolute changes in microdomain area over the thermal cycle. Error bars = 5% (see Methods).

Mentions: Interestingly, the microdomains showed a marked temperature dependence, whereas the overall dimensions of the membrane patches appeared unchanged over the range of temperatures probed in this work. This is illustrated in Fig 2, which shows the same membrane patches imaged over a temperature cycle between 25°C and the physiological 37°C. In particular, when the temperature was increased to 30°C, the smaller microdomains (less than 100 nm in diameter) disappeared while the larger ones were reduced in size, and the total area of the microdomains in this frame reduced by more than a factor 2. On a further temperature rise to 37°C, microdomains could hardly be discerned any more. As demonstrated by subsequent cooling to 25°C, this process was reversible in the sense that the total microdomain area was identical at the beginning and the end of the 25°C—30°C—37°C—25°C cycle, though the sizes and positions of individual microdomains had changed. This indicates a dynamic nature of the individual microdomain constituents at higher temperatures. At 37°C, the remaining microdomains could also be observed to gradually change shape and position within the membrane patches, without changing in size (S2 Fig). Further, prolonged incubation at temperatures > 37°C did not affect the reformation of microdomains on cooling below physiological temperature (S3 Fig).


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

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

Reversible dissolution and formation of microdomains.a, AFM topography of isolated membrane samples in buffer solution, for a thermal cycle in which the temperature is first increased from 25°C to 30°C and then 37°C, followed by a decrease back to 25°C. Arrows indicate the sequence of the images. Vertical (color) scale for all AFM images: 9 nm, see also scale bar in Fig 1. b, Histogram of the surface area of the microdomains observed on the membrane patches in (a), demonstrating a shrinking of microdomains for higher temperatures and a full recovery at the end of the thermal cycle (25°C*). c, Histogram of the total surface area of all patches that are fully included within the frames displayed in (a), showing that the absolute changes in total patch area are small compared to the absolute changes in microdomain area over the thermal cycle. Error bars = 5% (see Methods).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132696.g002: Reversible dissolution and formation of microdomains.a, AFM topography of isolated membrane samples in buffer solution, for a thermal cycle in which the temperature is first increased from 25°C to 30°C and then 37°C, followed by a decrease back to 25°C. Arrows indicate the sequence of the images. Vertical (color) scale for all AFM images: 9 nm, see also scale bar in Fig 1. b, Histogram of the surface area of the microdomains observed on the membrane patches in (a), demonstrating a shrinking of microdomains for higher temperatures and a full recovery at the end of the thermal cycle (25°C*). c, Histogram of the total surface area of all patches that are fully included within the frames displayed in (a), showing that the absolute changes in total patch area are small compared to the absolute changes in microdomain area over the thermal cycle. Error bars = 5% (see Methods).
Mentions: Interestingly, the microdomains showed a marked temperature dependence, whereas the overall dimensions of the membrane patches appeared unchanged over the range of temperatures probed in this work. This is illustrated in Fig 2, which shows the same membrane patches imaged over a temperature cycle between 25°C and the physiological 37°C. In particular, when the temperature was increased to 30°C, the smaller microdomains (less than 100 nm in diameter) disappeared while the larger ones were reduced in size, and the total area of the microdomains in this frame reduced by more than a factor 2. On a further temperature rise to 37°C, microdomains could hardly be discerned any more. As demonstrated by subsequent cooling to 25°C, this process was reversible in the sense that the total microdomain area was identical at the beginning and the end of the 25°C—30°C—37°C—25°C cycle, though the sizes and positions of individual microdomains had changed. This indicates a dynamic nature of the individual microdomain constituents at higher temperatures. At 37°C, the remaining microdomains could also be observed to gradually change shape and position within the membrane patches, without changing in size (S2 Fig). Further, prolonged incubation at temperatures > 37°C did not affect the reformation of microdomains on cooling below physiological temperature (S3 Fig).

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