Phase separation of giant unilamellar vesicles

Phase-separated giant unilamellar vesicles were produced by electroformation as described in Section 3.2.1. The vesicles were composed of the phospholipid DOPC, sphingomyelin, cholesterol and the Shiga toxin receptor lipid globotriaosylceramide (Gb₃) in a molar ratio of 40:35:20:5. In this chapter, Gb₃ from porcine erythrocytes (Gb₃-Porc) bearing different fatty acids was used (Section 3.1.3).

Figure 5.4A shows a fluorescence confocal slice of a GUV. The phase separation at room temperature intoldand lo domains was visualized by the enrichment of a lipid coupled fluorophor in the disordered phase. The bright green regions correspond told

domains. Darker regions on the circumference correspond to thel_o phase excluding the fluorophor.

A B

l

l

Figure 5.4: Fluorescence micrographs of a phase-separated GUV composed of DOPC/SM/Chol/Gb₃-Porc 40:35:20:5 doped with 0.5 mol% of TexasRed DHPE (false colored in green).A Confocal slice imaged at the equatorial position of the GUV. Bright green fluorescence depicts the enrichment of TexasRed in thel_d phase. Dark regions along the circular circumference correspond tol_odomains. Scale bar 10 µm.B3D reconstruction of a z-stack of the GUV shown inA.

In panel B a 3D reconstruction of the GUV is shown. The ld phase forms large domains with diameters of several micrometers on the surface of the GUV.^{42, 136} Due to the large size of the GUV (diameter ≈40 µm) the surface can be considered as fully flat on the molecular level.

Determination of the l_o percentage in GUVs

To understand the phase behavior of the lipid membrane, area percentages of thel_d andl_o phase were quantified as described in Section 3.4.1. Briefly, z-stacks of inflated GUVs were separated into two half spheres. Each was projected onto a plane using an orthographic azimuthal projection. The resulting projections were thresholded.

To correct for the area distortion of the projection the appropriate weighting

function was employed. Figure 5.5 shows the projection of a GUV composed of DOPC/SM/Chol/Gb₃-Porc (40:35:20:5) doped with 0.5 mol% TexasRed DHPE.

A B C D

Figure 5.5: Orthographic projections and thresholded images showing the l_d phase of a vesicle composed of DOPC/SM/Chol/Gb₃-Porc (40:35:20:5) doped with 0.5 mol%

TexasRed DHPE, imaged under hyperosmotic conditions in PBS. A Projection of the bottom cap of the GUV. The l_d phase is identified by the stronger fluorescence intensity.

B Thresholded image of A. White areas correspond to the l_d phase.CProjection of the upper cap of the GUV. Identification of thel_dphase by the stronger fluorescence intensity is possible. B Thresholded image of C. Scale bars: 10 µm.

The projections presented in Figure 5.5A/C were manually thresholded. Pixel analysis of the thresholded images compared to the total area of the projection yielded the l_o phase percentage which was corrected for the area distortion of the projection.

Analysis of 112 GUVs with a mean diameter of 15±9 µm gave a l_o percentage of 36±10 % in good agreement with values reported in literature.^{37, 115} As the distribution of the lipid domains in a non adhering GUV is random, the correction for the area distortion did not alter the l_o percentage compared to the uncorrected case. Three different lipid dyes (Bodipy-PC, Atto488 DHPE, TexasRed DHPE) were employed and the percentage was found to be independent of the dye used.

Shiga toxin binding to Gb3 containing GUVs

STxB binding to Gb₃ is known to alter the membrane organization.70, 71, 86, 134 The area of the l_o phase of the GUVs after STxB binding was quantified. GUVs were incubated with STxB or Cy3-labeled STxB (STxB-Cy3) at a final concentration of 60 nm. Figure 5.6 shows the projections of a GUV after STxB-Cy3 incubation for 1 h.

A B

Figure 5.6:Orthographic projections of a GUV composed of DOPC/SM/Chol/Gb₃-Porc (40:35:20:5) doped with 0.5 mol% Bodipy-PC imaged under hyperosmotic conditions in PBS after incubation with STxB-Cy3.A Projection of the bottom cap of the GUV. The l_d phase is identified by the stronger green Bodipy fluorescence intensity. The l_o phase is stained red by STxB-Cy3 binding to Gb₃-Porc. In the right part several adhered vesicles are imaged.B Projection of the upper cap of the GUV. Scale bars: 10 µm.

The red fluorescence shows that STxB binds to Gb₃-Porc in the giant vesicles. The fluorescence signal of STxB-Cy3 is only located on the l_o domain.⁷⁰ l_d domains can again be identified by the fluorescence of the l_d marker dye.

To elucidate if STxB binding changes the lateral organization by lipid redistribution between the phases, the area percentage of the lo phase was determined by thresholding fluorescence micrographs of the l_d marker dye. Figure 5.7 shows an exemplary GUV after STxB binding.

A B C D

Figure 5.7: Orthographic projections and thresholded images showing the l_d phase of a vesicle composed of DOPC/SM/Chol/Gb₃-Porc (40:35:20:5) doped with 0.5 mol%

TexasRed DHPE, imaged under hyperosmotic conditions in PBS after incubation with STxB-Cy3.A Projection of the bottom cap of the GUV. Thel_d phase is identified by the stronger fluorescence intensity. B Thresholded image of A. The white areas correspond to thel_d phase.CProjection of the upper cap of the GUV. The l_d phase is identified by the stronger fluorescence intensity of thel_d marker dye TexasRed DHPE.B Thresholded image of C. Some small domains were not properly detected during thresholding. Scale bars: 8 µm.

The analysis of 99 GUVs with a mean diameter of 14±6 µm gave a l_o percentage of 34±12 %. Three different lipid dyes (Bodipy-PC, Atto488 DHPE, TexasRed DHPE) were used and the percentage was found to be independent of the dye.

Determination of l_o area percentage after spreading of GUVs

To test whether a solid support has an influence on the phase separation, GUVs were spread on silicon dioxide wafers above the phase transition temperature as described in Section 3.4.1. Figure 5.8 shows characteristic fluorescence micrographs obtained after spreading.

D E F

A B C

Figure 5.8: Fluorescence micrographs of phase-separated GUVs composed of DOPC/SM/Chol/Gb₃-Porc (40:35:20:5) doped with 0.5 mol% TexasRed DHPE spread on silicon dioxide wafers. Top row: Imaged prior to protein binding. Bottom row: Imaged after incubation with STxB. A/D Fluorescence micrographs of TexasRed show the two different lipids phases.B/ETotal areas of the membrane patches.C/FRegions identified as the l_d phase. Scale bars: 10 µm.

The substrate is not fully covered with membrane but isolated patches that correspond to the lipid material of single GUVs are found.^{45, 201} The total area of the patch was determined and compared to the area of the l_d phase obtained from thresholding the fluorescence micrographs. The total area was used to calculate the diameter of the GUV that was spread on the surface. In absence of bound STxB

the l_o percentage was determined to be 38±11 % (n = 22). After STxB binding the area percentage was 38±8 % (n = 21), showing that the areas of the phases are the same as in the GUV.

Table 5.1 summarizes the results obtained for thel_opercentages in GUVs. Correction for the area distortion did not alter the l_o percentage compared to the uncorrected case.

Table 5.1: Summary of the l_o area percentages determined in GUVs and in membranes obtained from GUV spreading. For the latter the radius was calculated from the area of the obtained membrane patch.

% l_o phase GUV diameter / µm n

GUVs –STxB 36±10 15±9 112

+STxB 34±12 14±6 99

spread –STxB 38±11 12±4 22

GUVs +STxB 38±8 16±6 21

Binding of STxB to the membrane lead to no alteration of thel_o phase percentage.

This indicates that no major rearrangement of lipid between the phases took place.