Content mixing of LUVs with adhered GUVs

The ultimate proof for imaging of two bilayers in the context of vesicle-vesicle fusion is content mixing.

The content mixing experiment, illustrated in figure 5.7, was done by adding LUVs, containing the water soluble dye ATTO^® 488, to the sample with the adhered GUVs. An increased fluorescence intensity inside the GUV reveals that the LUVs fuse fully with the GUV-membrane. Merging of vesicle membranes is mediated by the use of SNARE proteins on both vesicles. The LUVs contained synaptobrevin, and the GUVs the ΔN49-complex.

In figure 5.15 three GUVs with different sizes and adhesion areas are shown in a xy-plane picture and a corresponding cross-sectional view on the right and bottom. After the incubation of LUVs containing the water soluble green fluorescent dye at the inside of the LUVs it was detected that the GUVs 27 and 28 show a high fluorescence intensity compared to GUV 26 with nearly no intensity emitted from the inside (figure 5.15 C+D). The result for the calculated membrane tension on each GUV and the corresponding green fluorescence intensity inside the GUV is given in the table 5.1. The surrounding solution served as a reference for the fluorescence intensity I488 inside the GUVs.

A small adhesion area of GUV 26 generated a low membrane tension of 0.12 mN/m. The larger adhesion area compared to the vesicle radius on GUV 27 and 28 resulted in a larger membrane tension of 0.29 mN/m for GUV 27 and 0.94 mN/m for GUV 28. Interestingly, the fluorescence intensity originating from the LUV content dye ATTO^® 488 increased inside the GUVs 27 and 28 up to 60% and respectively 100% of the fluorescence intensity in the surrounding solution whereas GUV 26 with a low membrane tension showed no significant fluorescence intensity increase (Table 5.1). The smallest GUV28 with the highest membrane tension contained the same fluorescence intensity inside the GUV as the surrounding buffer solution.

Figure 5.14. Mixing of contents was achieved by the fusion of LUVs to the adhered GUV so that the inside of the LUVs mixed with the GUV solution inside and the green fluorescent dye diffused into the GUV. Thus the green fluorescence intensity inside the GUV should increase.

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xy yz

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27

27

28 28

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C D

26 27

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26

Figure 5.15. Three GUVs slightly adhered on the glass surface of the sample. A) Three GUVs are depicted in on image xy-image plane. B) The cross-sectional image for GUV 26 is shown. C) After LUV incubation GUVs 27 and 28 contained the green fluorescent dye ATTO^® 488. D) The largest GUV with the smallest membrane tension shows no fluorescence intensity from the LUV dye

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Table 5.1. The contact radius Ri and the vesicle radius R̃_v was measured for each GUV. With these measured values for each GUV the area change ΔA/A0 and the corresponding membrane tension was calculated that can be compared to the fluorescence intensity of the LUV content dye that was measured at the inside of the GUV after LUV fusion possibly occurred. A comparison of the LUV volume to the GUV volume reveals how many time the GUVs are greater than an average LUV with a diameter of 200 nm.

GUV No. R_i / µm R̃_v / µm ΔA/A0 / % 𝜏 / mN m^-1 I488 / % V̅_LUV / V_GUV

26 4.30 13.98 0.05 0.12 6.6 1 / (2.7 × 10⁶)

27 3.35 7.31 0.28 0.29 60 1 / (3.9 × 10⁵)

28 2.88 5.01 0.73 0.94 100 1 / (1.3 × 10⁵)

The three GUVs 26-28 in figure 5.15 contained different volumes. Therefore, the dilution of the dissolved LUV content dye could affect the measurement for the fluorescence intensity inside the GUVs. The difference in dilution between the GUVs can be compared by the LUV-volume V̅_LUV with the three the GUV-volumes V_GUV. The mean size of the LUVs can be assumed to be around 200 nm in diameter containing a mean volume V̅_LUV = 4.2 × 10^-3 µm³. The mean volume V̅_LUV of the LUVs is compared to the three GUVs 26-28 in table 5.1. The largest GUV 26 compared to the smallest GUV 28 contains a volume that is one order of magnitude larger which implies that the dilution could play a central role to the measured fluorescence intensity at the inside of each GUV.

A FRAP-measurement, shown in figure 5.16, shows that the GUV content was bleached by the LASER and thus, content mixing of LUVs to the GUV 28 was proven. The fluorescence intensity in the GUV before the bleaching with the LASER was lower compared to figure 5.15 because of the scanning LASER of the microscope. However, the graph in figure 5.16 D plots the fluorescence intensity inside

A B C

D

Figure 5.16. FRAP measurement of GUV 3 with on a longer time scale. The images were taken before (A) and after (B) bleaching of the GUV content at 6 s. Image (C) was the last one 324 s after the GUV content was bleached. D) The graph outlines the green fluorescence intensity inside the GUV and in the buffer solution as a reference. A slightly increase of the green dye in the GUV was measured during time which levels off at around 160 s at an intensity of 0.22 a.u.

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the GUV as a function of time from the FRAP-measurement. Interestingly, the fluorescence intensity slightly increased after the bleaching which indicates that LUVs release their content into the GUV through membrane fusion. To exclude a diffusion of the dye ATTO^® 488 from the surrounding buffer solution into the GUV a blind sample without SNAREs in figure 5.17 is depicted. Even strongly adhered GUVs with a high membrane tension do not contain any fluorescence intensity so that a diffusion of the water soluble LUV dye through the GUV-membrane is disproved.

For the two GUVs 29 and 30 from figure 5.17 the calculated membrane tension is listed in the table (B).

In this blind sample no content mixing was detected although the membrane tensions of the GUVs occurred to be much higher compared to the GUVs 27 and 28.

Summing up the results of this chapter, the content mixing experiment proved that full fusion of the two lipid bilayers between LUVs and tensed GUVs is achieved by using SNAREs as fusion promoters.

A

30

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GUV No. ^Ri / µm R̃_v / µm ΔA/A0 / % 𝜏 / mN m^-1

29 5.7 7.2 3.2 6.9

30 4.0 5.9 1.4 2.3

Figure 5.17. A) A blind sample without SNAREs shows GUVs after incubation of LUVs but without any diffusion of the green fluorescent dye into the content of the GUVs. LUV fusion or diffusion of the green dye from the buffer solution into the GUVs did not occur. The cross-sectional view shows that the GUVs strongly adhere on the surface. B) The table lists the measure radii, area changes and the calculated membrane tension of the GUVs 29 and 30.

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10 µm

B

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