Visualization of detergent solubilization of membranes: implications for the isolation of rafts

Abstract

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (l(d)) and raft liquid ordered (l(o)) lipid phases by selectively solubilizing the l(d) phase. A higher concentration of Lubrol was required, and not all the l(d) phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l(d) phase and then progressed to the solubilization of both l(d) and l(o) phases simultaneously. Octyl glucoside simultaneously solubilized both l(o) and l(d) phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.

FIGURE 1

Solubilization of SLBs and CHO cells by TX100. (A) AFM images of SLBs at five time points over 60 min after the addition of 0.015%, 0.045%, 0.075%, or 0.1% TX100. At 0.015% TX100, no solubilization of the SLB occurs. Solubilization of the l_d phase begins after 5 min at 0.045% TX100, as indicated by the dark regions in the AFM image. After 30 min at 0.045%, solubilization of the l_d phase is complete but the l_o phase remains TX100 resistant. At 0.075% and 0.1% TX100, complete solubilization of the l_d phase occurs within 5 min, and the l_o domains are still resistant to solubilization after 60 min. Images are 10 μm scans with 10 nm height scale. (B) The surface area in the l_o phase as a percentage of the total area was determined for each AFM image after the addition of the indicated concentration of TX100 to SLBs. (C) CHO cells coexpressing GPI-CFP and VSVG-YFP were imaged by confocal microscopy every 4 s for 3 min during solubilization by 0.05% TX100. In contrast to the rapidly solubilized VSVG-YFP, GPI-CFP exhibited relative resistance to TX100 solubilization. Bar = 10 μm. (D) Percentage of fluorescence from GPI-CFP and VSVG-YFP during solubilization of CHO cells with TX100 as in (C).

FIGURE 2

Solubilization of SLBs and CHO cells by Lubrol. (A) AFM images of SLBs at five time points over 60 min after the addition of 0.008%, 0.04%, 0.1%, or 0.4% Lubrol. No solubilization of the SLBs was observed at 0.008% or 0.04%, and only small holes began to form in the l_d phase at 0.1% Lubrol. The addition of 0.4% Lubrol resulted in extensive solubilization which predominately occurred at the interface between the l_d and l_o phases. Images are 10 μm scans with 10 nm height scale. (B) The surface area in the l_o phase as a percentage of the total area was determined for each AFM image after the addition of the indicated concentration of Lubrol to SLBs. (C) CHO cells coexpressing GPI-CFP and VSVG-YFP were imaged by confocal microscopy every 4 s for 3 min after the addition of 0.45% Lubrol. VSVG-YFP was preferentially solubilized initially, with GPI-CFP solubilization accompanying the loss of membrane integrity. Bar = 10 μm. (D) Percentage of fluorescence from GPI-CFP and VSVG-YFP during solubilization of CHO cells by 0.45% Lubrol as in (C).

FIGURE 3

Solubilization of SLBs and CHO cells by Brij 96. (A) AFM images of SLBs at five time points over 60 min after the addition of 0.029%, 0.087%, 0.1%, or 0.145% Brij 96. Solubilization of the SLBs was observed at all concentrations and although holes originated in the l_d phase, progressive solubilization appears to occur at the interface between the l_d and l_o phase. Images are 10 μm scans with 10 nm height scale. (B) The surface area in the l_o phase as a percentage of the total area was determined for each AFM image after the addition of the indicated concentration of Brij 96 to SLBs. (C) CHO cells coexpressing GPI-CFP and VSVG-YFP were imaged by confocal microscopy every 4 s for 3 min after the addition of 0.075% Brij 96. Preferential solubilization of VSVG-YFP in the first minute was followed by GPI-CFP solubilization. A small proportion of both proteins remained after 3 min. Bar = 10 μm. (D) Percentage of fluorescence from GPI-CFP and VSVG-YFP during solubilization of CHO cells with 0.075% Brij 96 as in C.

FIGURE 4

Solubilization of SLBs and CHO cells by CHAPS. (A) AFM images of SLBs at five time points over 60 min after the addition of 0.1%, 0.37%, 1.11%, or 1.85% CHAPS. No solubilization of the SLB was observed at 0.1%. Complete solubilization of the l_d phase was observed at 0.37%, whereas the l_o domains remained CHAPS resistant. At 1.11% CHAPS solubilization of the l_d phase was followed by progressive l_o solubilization, and at 1.85% CHAPS the whole SLB was solubilized within 30 min. Images are 10 μm scans with 10 nm height scale. (B) The surface area in the l_o phase as a percentage of the total area was determined for each AFM image after the addition of the indicated concentration of CHAPS to SLBs. (C) CHO cells coexpressing GPI-CFP and VSVG-YFP were imaged by confocal microscopy every 4 s for 3 min after the addition of 0.5% CHAPS. Significant loss of both proteins was observed, but GPI-CFP was comparatively more resistant to CHAPS solubilization. Bar = 10 μm. (D) Percentage of fluorescence from GPI-CFP and VSVG-YFP during solubilization of CHO cells with 0.5% CHAPS as in (C).

FIGURE 5

Solubilization of SLBs and CHO cells by OG. (A) AFM images of SLBs at five time points are shown after the addition of 0.1%, 0.7%, 2.1%, or 3.5% OG. Some small holes formed in the SLB at 0.1% OG but little solubilization occurred. All but two lipid domains were solubilized at 0.7% OG, and the whole SLB was solubilized within 5 min at 2.1% and 3.5% OG. Images are 10 μm scans with 10 nm height scale. (B) The surface area in the l_o phase as a percentage of the total area was determined for each AFM image after the addition of the indicated concentration of OG to SLBs. (C) CHO cells coexpressing GPI-CFP and VSVG-YFP were imaged by confocal microscopy every 4 s for 3 min after the addition of 0.75% OG. Considerable loss of both proteins from the cell surface was observed within 1 min. The bright fluorescent spot in subsequent images is contamination from a dead cell. Bar = 10 μm. (D) Percentage of fluorescence from GPI-CFP and VSVG-YFP during solubilization of CHO cells with 0.75% OG as in (C). To reflect the relative fluorescence of the live cells only, the bright spot of fluorescence observed in (C) was excluded.