AFM Characterization of Model Rafts in Supported Bilayers

Abstract

Microdomains formed by a phase separation of lipids play an important role in the function of biological membranes. GM1 is a glycosphingolipid associated with rafts, a category of membrane domains. Whereas GM1 has been shown to form microdomains in Langmuir-Blodgett films its behaviour in lipid bilayers is still under debate. In the present study we show by atomic force microscopy that GM1 can form microdomains in supported bilayers. These microdomains are exclusively found in the gel domains of a gel-fluid phase separated mixture of phospholipids. The localization of GM1 in gel domains was confirmed by the use of cholera toxin which binds to the oligosaccharide moiety of the glycosphingolipid. Finally, the data also show that imaging of proteins loosely packed at the membrane surface can be obtained with a lateral resolution close to 1 nm.

The Size of Lipid Rafts: An Atomic Force Microscopy Study of Ganglioside GM1 Domains in Sphingomyelin/DOPC/Cholesterol Membranes

“…As noted above the present results are analogous to earlier studies that showed similar small GM1-rich domains in PC and PC/cholesterol monolayers and bilayers (Milhiet et al, 2001b;Vie et al, 1998;Johnston, 2000, 2001). In fact the potential for glycosphingolipids to form domains has been known for some time (Thompson et al, 1985).…”

The Size of Lipid Rafts: An Atomic Force Microscopy Study of Ganglioside GM1 Domains in Sphingomyelin/DOPC/Cholesterol Membranes

“…As noted above the present results are analogous to earlier studies that showed similar small GM1-rich domains in PC and PC/cholesterol monolayers and bilayers (Milhiet et al, 2001b;Vie et al, 1998;Johnston, 2000, 2001). In fact the potential for glycosphingolipids to form domains has been known for some time (Thompson et al, 1985).…”

Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study.

“…2A-D). This is again expected but was not found from previous AFM studies which used larger protein concentration (Milhiet et al, 2002a;2002b;Saslowsky et al, 2002). It is interesting to note that incubation in 1.6 mg/ml AP GPI (Fig.…”

“…2C is in accordance with a pure DPPC bilayer with a protein layer above. When they are adsorbed directly on bare substrates, single or aggregated proteins protrude by 3 to 5 nm (Rieu et al, 2002) while the incubation of AP with supported mixed bilayers, resulted in the apparition of dots protruding by 2 to 5 nm from the membrane (Milhiet et al, 2002a;2002b). At smaller scales, this bilayer shows a granular structure with a shorter characteristic grain diameter than before incubation, i.e., 10-15 nm, a larger peak to valley height, i.e., 2 nm, and a larger rms roughness, i.e., 0.4 nm (Fig.…”

“…The aim of this work was to determine the spatial organization of inserted GPI-anchored alkaline phosphatase within supported phospholipids bilayers. Several recent studies demonstrated that GPI proteins may be spontaneously incorporated into supported lipid bilayers and individually detected by AFM (Milhiet et al, 2002a;2002b, Saslowsky et al, 2002. In these studies, bilayers were incubated in the presence of 30-50 mg/ml of protein.…”

Observation of the formation of supported bilayers by amphiphilic peptidyl-RNA

“…AFM has allowed nanometric resolution imaging of the in situ formation of SLBs, the formation of rafts in SLBs and especially the interactions between biological macromolecules and SLBs. [5][6][7] In the present communication we report on the observation of the formation of thick (about 21 nm) supported bilayers by peptidyl-RNA molecules on optical glass surfaces. 8 The structure and a model of the peptidyl-RNA molecule, Ala 21 RNA 22 , used in this study is presented in Fig.…”

The Size of Lipid Rafts: An Atomic Force Microscopy Study of Ganglioside GM1 Domains in Sphingomyelin/DOPC/Cholesterol Membranes

“…As noted above the present results are analogous to earlier studies that showed similar small GM1-rich domains in PC and PC/cholesterol monolayers and bilayers (Milhiet et al, 2001b;Vie et al, 1998;Johnston, 2000, 2001). In fact the potential for glycosphingolipids to form domains has been known for some time (Thompson et al, 1985).…”

Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study.

“…2A-D). This is again expected but was not found from previous AFM studies which used larger protein concentration (Milhiet et al, 2002a;2002b;Saslowsky et al, 2002). It is interesting to note that incubation in 1.6 mg/ml AP GPI (Fig.…”

“…2C is in accordance with a pure DPPC bilayer with a protein layer above. When they are adsorbed directly on bare substrates, single or aggregated proteins protrude by 3 to 5 nm (Rieu et al, 2002) while the incubation of AP with supported mixed bilayers, resulted in the apparition of dots protruding by 2 to 5 nm from the membrane (Milhiet et al, 2002a;2002b). At smaller scales, this bilayer shows a granular structure with a shorter characteristic grain diameter than before incubation, i.e., 10-15 nm, a larger peak to valley height, i.e., 2 nm, and a larger rms roughness, i.e., 0.4 nm (Fig.…”

“…The aim of this work was to determine the spatial organization of inserted GPI-anchored alkaline phosphatase within supported phospholipids bilayers. Several recent studies demonstrated that GPI proteins may be spontaneously incorporated into supported lipid bilayers and individually detected by AFM (Milhiet et al, 2002a;2002b, Saslowsky et al, 2002. In these studies, bilayers were incubated in the presence of 30-50 mg/ml of protein.…”

Observation of the formation of supported bilayers by amphiphilic peptidyl-RNA

“…AFM has allowed nanometric resolution imaging of the in situ formation of SLBs, the formation of rafts in SLBs and especially the interactions between biological macromolecules and SLBs. [5][6][7] In the present communication we report on the observation of the formation of thick (about 21 nm) supported bilayers by peptidyl-RNA molecules on optical glass surfaces. 8 The structure and a model of the peptidyl-RNA molecule, Ala 21 RNA 22 , used in this study is presented in Fig.…”

The Size of Lipid Rafts: An Atomic Force Microscopy Study of Ganglioside GM1 Domains in Sphingomyelin/DOPC/Cholesterol Membranes

“…As noted above the present results are analogous to earlier studies that showed similar small GM1-rich domains in PC and PC/cholesterol monolayers and bilayers (Milhiet et al, 2001b;Vie et al, 1998;Johnston, 2000, 2001). In fact the potential for glycosphingolipids to form domains has been known for some time (Thompson et al, 1985).…”

Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study.

“…2A-D). This is again expected but was not found from previous AFM studies which used larger protein concentration (Milhiet et al, 2002a;2002b;Saslowsky et al, 2002). It is interesting to note that incubation in 1.6 mg/ml AP GPI (Fig.…”

“…2C is in accordance with a pure DPPC bilayer with a protein layer above. When they are adsorbed directly on bare substrates, single or aggregated proteins protrude by 3 to 5 nm (Rieu et al, 2002) while the incubation of AP with supported mixed bilayers, resulted in the apparition of dots protruding by 2 to 5 nm from the membrane (Milhiet et al, 2002a;2002b). At smaller scales, this bilayer shows a granular structure with a shorter characteristic grain diameter than before incubation, i.e., 10-15 nm, a larger peak to valley height, i.e., 2 nm, and a larger rms roughness, i.e., 0.4 nm (Fig.…”

“…The aim of this work was to determine the spatial organization of inserted GPI-anchored alkaline phosphatase within supported phospholipids bilayers. Several recent studies demonstrated that GPI proteins may be spontaneously incorporated into supported lipid bilayers and individually detected by AFM (Milhiet et al, 2002a;2002b, Saslowsky et al, 2002. In these studies, bilayers were incubated in the presence of 30-50 mg/ml of protein.…”

Observation of the formation of supported bilayers by amphiphilic peptidyl-RNA

“…AFM has allowed nanometric resolution imaging of the in situ formation of SLBs, the formation of rafts in SLBs and especially the interactions between biological macromolecules and SLBs. [5][6][7] In the present communication we report on the observation of the formation of thick (about 21 nm) supported bilayers by peptidyl-RNA molecules on optical glass surfaces. 8 The structure and a model of the peptidyl-RNA molecule, Ala 21 RNA 22 , used in this study is presented in Fig.…”