Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors

Abstract

We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of giant unilamellar vesicles and a supported lipid bilayer, triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs. We confirmed that almost all newly exchanged lipids in the aGUVs are found in the outer leaflet of these asymmetric vesicles. In addition, we test the stability of the aGUVs formed by hemifusion in preserving their contents during the procedure. For aGUVs prepared from the hemifusion of giant unilamellar vesicles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.39/0.39/0.22 and a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.8/0.2, we observed the exchanged lipids to alter the bilayer properties. To access the physical and chemical properties of the asymmetric bilayer, we monitored the dye partition coefficients of individual leaflets and the generalized polarization of the fluorescence probe 6-dodecanoyl-2-[ N-methyl-N-(carboxymethyl)amino] naphthalene, a sensor for the lipid packing/order of its surroundings. For a high percentage of lipid exchange (>70%), the dye partition indicates induced-disordered and induced-ordered domains. The induced domains have distinct lipid packing/order compared to the symmetric liquid-disordered and liquid-ordered domains.

Figure 1

Experimental procedure and analyses. (A) A sketch of the hemifusion between a supported lipid bilayer (SLB) and a symmetric GUV is shown. GUVs and SLB were prepared with different fluorescent labels represented by different colored lipids. After hemifusion, aGUVs are detached from the SLB. (B) A comparison of symmetric and aGUVs is shown. Examples of symmetric GUVs are shown as follows (from left to right): control GUV 1 + red DiD, control GUV 2 + green TFPC, and example of an aGUV; the image shows the red, green, and merged channels of the microscope for one aGUV. Line scans are displayed in white, indicating θ = 0. (C) Intensity profiles of the line scans shown on (B), where the intensity peak on the vesicle radius R is measured. The left panel shows a comparison between the intensity of DiD for symmetric and aGUV; the right panel shows a comparison between the intensity of TFPC for symmetric and aGUV. Lipids and TFPC of the SLB replace the lipids and DiD of the GUV. (D) The intensity measured on the GUV or aGUV radius as a function of θ is shown. To see this figure in color, go online.

Figure 2

Microscopy images reveal the hemifusion process. (A) Shown is a hemifusion between a supported lipid bilayer (SLB) of DOPC + TFPC (green) and a GUV of DSPC/DOPC/chol + DiD (red), forming an aGUV that displays both dyes. (B) The SLB immediately after detaching the aGUV is shown. The following are shown from left to right: a three-dimensional view of the microscope red channel, which reveals DiD fluorescence; a three-dimensional view of the microscope green channel, which reveals TFPC fluorescence and red and green merge channels. To see this figure in color, go online.

Figure 3

Percentage of lipids exchanged after hemifusion. aGUVs prepared from the hemifusion of GUVs labeled with DiD and an SLB labeled with TFPC are shown. (A) Bar plots represent the intensity levels observed in symmetric (solid bar). Dots on the side of solid bars represent the measurements of different symmetric GUVs for which the average intensity was calculated from 360 line scans (see text). Open bars show different percentage of exchanged lipids (right axis) calculated by the comparison of intensities in symmetric and asymmetric vesicles. Errors were calculated from the standard deviation of 360 intensity values for each dye. (B) The percentage of exchanged lipids was independently calculated for DiD and TFPC, [Ca²⁺] = 4–7 mM. Line of slope 1 shows the agreement of the calculated percentage of lipid exchange for DiD and TFPC. Errors were calculated by propagating the uncertainty obtained in the fluorescence signal. To see this figure in color, go online.

Figure 4

Quenching assay of LUVs using sodium dithionite. (A) Normalized fluorescence (arb. u.) as a function of time for 100 nm LUVs prepared with a single dye is shown: NBD-PE (solid square), DiD (solid circle), or TFPC (open diamond). (B) Normalized light scattering of LUVs simultaneously recorded as a function of time is shown. Arrow 1 indicates the time at which dithionite was added. Arrow 2 indicates the addition of Triton X-100 (10% w/v). Dithionite efficiently quenches NBD-PE and DiD. To see this figure in color, go online.

Figure 5

Fluorescence quenching of an aGUV outer leaflet confirms hemifusion. (A) aGUVs prepared from the hemifusion of GUVs labeled with DiD and an SLB labeled with NBD-PE are shown. The percentage of exchanged lipids was independently calculated for DiD and NBD-PE (see text; [Ca²⁺] = 5–8 mM). Errors were calculated by propagating the uncertainty obtained in the fluorescence signal. (B) After the addition of dithionite, the fluorescence of DiD and NBD-PE in the outer leaflet is quenched. The normalized fluorescence of DiD is reduced to half of the value of the unquenched case (left) (n = 21). For a sample of 21 aGUVs, only 5 aGUVs display fluorescence signals of NBD-PE after quenching, with an averaged normalized intensity equivalent to 4% of the value of the unquenched case. Errors were calculated from the standard deviation of 360 intensity values for each dye. The exchanged lipids that come from the SLB are mainly found on the aGUV outer leaflet. To see this figure in color, go online.

Figure 6

Fraction of aGUVs created and aGUV content release. (A) A symmetric GUV prepared with trapped carboxyfluorescein (CF) dye without membrane dye is shown. (B) An aGUV prepared by hemifusion between symmetric GUV as displayed in (A) and SLB labeled with red DiD is shown. (C) Fully fused GUV that has released the trapped dye is shown. (D) The following are shown (from left to right): the fraction of symmetric GUVs that did not interact with the SLB, aGUVs with preserved contents, aGUVs with slight leakage of the trapped dye, and fully fused GUVs obtained after the hemifusion procedure (n = 114). To see this figure in color, go online.

Figure 7

Changes in physical chemical properties of aGUVs. (A) Phase diagram of DSPC/DOPC/chol. The lipid composition of the symmetric GUVs and the inner leaflet of aGUVs are described by the star (middle point of tieline A). aGUVs prepared from the hemifusion of GUVs composed of DSPC/DOPC/chol = 0.39/0.39/0.22 + DiD and an SLB composed of DOPC/chol = 0.8/0.2 + TFPC are shown. For aGUVs, the lipid composition of the outer leaflet follows the trajectory described by the arrow as the percentage of lipid exchange increases. (B) The aGUVs result from lipid exchange between GUVs that hemifused with the SLB. The percentage of exchanged lipids was independently calculated for DiD and TFPC (see text; [Ca²⁺] = 5–8 mM). (C) The apparent partition coefficient, Kp^app, of TFPC as a function of percentage of exchanged lipids is shown. TFPC labels the outer leaflet. (D) The partition coefficient of DiD as a function of exchanged lipids after quenching by dithionite is shown. The Kp of DiD refers to the inner leaflet. Error bars shown in (B)–(D) were calculated by propagating the uncertainty obtained in the fluorescence signal. To see this figure in color, go online.

Figure 8

C-Laurdan reports on lipid packing in symmetric and aGUVs. (A) Spectral images of symmetric and (B) aGUVs using C-Laurdan are shown. (C) A color map of GP values of (left) a symmetric GUV and (right) an aGUV is shown. (D) A comparison between blue-shifted spectra, indicating ordered domains, for symmetric GUVs and aGUVs is shown. (E) A comparison between red-shifted spectra, indicating disordered domains, for symmetric and aGUVs is shown. (F) Frequency counting of GP values measured on the GUV and aGUV is shown. aGUVs, with the outer leaflet enriched in DOPC, display considerable difference in the lipid packing/order compared to the symmetric GUV. The percentage of lipid exchange for this aGUV is ∼85 ± 3% (B and C). Scale bars, 10 μm (A and B). ΔGP = <GP>(ordered domains) − <GP>(disordered domains). To see this figure in color, go online.