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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

Immunolabelling of microdomains.AFM at room temperature to detect protein content in the microdomains, before (left) and after (right) a thermal cycle (25°C -37°C- 40°C- 25°C). a, Untreated membrane patches were incubated with anti flotillin-1 antibodies. b, After 60 min of incubation, the area of the microdomains protruding from the membrane patches had increased (see differently colored circles for individual examples). c, Histogram of the surface area increase for membrane patches and microdomains after 60 min antibody incubation. d, e, f, As (a, b, c), but for membrane patches that have undergone a thermal cycle prior to the immunolabeling. Student's t-test for the difference between relative increases of patch and microdomain areas: p<0.01 in both (c) and (f). Horizontal scale bar: 1 μm; vertical (color) scale: 9 nm in the four AFM images.
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pone.0132696.g004: Immunolabelling of microdomains.AFM at room temperature to detect protein content in the microdomains, before (left) and after (right) a thermal cycle (25°C -37°C- 40°C- 25°C). a, Untreated membrane patches were incubated with anti flotillin-1 antibodies. b, After 60 min of incubation, the area of the microdomains protruding from the membrane patches had increased (see differently colored circles for individual examples). c, Histogram of the surface area increase for membrane patches and microdomains after 60 min antibody incubation. d, e, f, As (a, b, c), but for membrane patches that have undergone a thermal cycle prior to the immunolabeling. Student's t-test for the difference between relative increases of patch and microdomain areas: p<0.01 in both (c) and (f). Horizontal scale bar: 1 μm; vertical (color) scale: 9 nm in the four AFM images.

Mentions: To verify for protein content in the observed microdomains, the purified and adsorbed membrane samples were imaged before and after 60min incubation with 50 μg/ml antibodies to target flotillin-1, a protein associated with lipid rafts [33], at room temperature. As observed previously [31], this immunolabeling lead to a relative increase of the microdomain areas that is significantly larger than that of the overall membrane patches (Fig 4A–4C). Importantly, this increase was the same irrespective of whether the immunolabeling was performed before or after a thermal cycle in which the temperature had been raised to 40°C (thermal cycle 25°C-37°C-40°C-25°C; Fig 4D–4F). This demonstrates that the protein content of the microdomains is recovered after dissolution and reformation of the microdomains, and thus confirms the overall reversibility of the temperature-induced changes in the microdomains.


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

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

Immunolabelling of microdomains.AFM at room temperature to detect protein content in the microdomains, before (left) and after (right) a thermal cycle (25°C -37°C- 40°C- 25°C). a, Untreated membrane patches were incubated with anti flotillin-1 antibodies. b, After 60 min of incubation, the area of the microdomains protruding from the membrane patches had increased (see differently colored circles for individual examples). c, Histogram of the surface area increase for membrane patches and microdomains after 60 min antibody incubation. d, e, f, As (a, b, c), but for membrane patches that have undergone a thermal cycle prior to the immunolabeling. Student's t-test for the difference between relative increases of patch and microdomain areas: p<0.01 in both (c) and (f). Horizontal scale bar: 1 μm; vertical (color) scale: 9 nm in the four AFM images.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132696.g004: Immunolabelling of microdomains.AFM at room temperature to detect protein content in the microdomains, before (left) and after (right) a thermal cycle (25°C -37°C- 40°C- 25°C). a, Untreated membrane patches were incubated with anti flotillin-1 antibodies. b, After 60 min of incubation, the area of the microdomains protruding from the membrane patches had increased (see differently colored circles for individual examples). c, Histogram of the surface area increase for membrane patches and microdomains after 60 min antibody incubation. d, e, f, As (a, b, c), but for membrane patches that have undergone a thermal cycle prior to the immunolabeling. Student's t-test for the difference between relative increases of patch and microdomain areas: p<0.01 in both (c) and (f). Horizontal scale bar: 1 μm; vertical (color) scale: 9 nm in the four AFM images.
Mentions: To verify for protein content in the observed microdomains, the purified and adsorbed membrane samples were imaged before and after 60min incubation with 50 μg/ml antibodies to target flotillin-1, a protein associated with lipid rafts [33], at room temperature. As observed previously [31], this immunolabeling lead to a relative increase of the microdomain areas that is significantly larger than that of the overall membrane patches (Fig 4A–4C). Importantly, this increase was the same irrespective of whether the immunolabeling was performed before or after a thermal cycle in which the temperature had been raised to 40°C (thermal cycle 25°C-37°C-40°C-25°C; Fig 4D–4F). This demonstrates that the protein content of the microdomains is recovered after dissolution and reformation of the microdomains, and thus confirms the overall reversibility of the temperature-induced changes in the microdomains.

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