Fusion of vesicles with pore-spanning membranes

Fusion of 90 nm and 240 nm syb 2 vesicles with the PSM was analyzed in a time resolved manner. As mentioned before, on the f-PSM only large 240 nm vesicles could be analyzed while on the s-PSM also the smaller 90 nm vesicles were detectable. Fusion events on the f-PSM could only very rarely be observed, as 89 % of the vesicles that initially docked to the f-PSM (N = 106) were immobilized on the pore rim between the s-PSM and f-PSM after some time (Chapter 6.3). As most of these immobilized vesicles could not regain their mobility, they were consequently analyzed as s-PSM fusion events. This feature and the 50 % lower docking probability on the f-PSM (Chapter 6.4.2) explains why in total only 3 % of the docked vesicles (N = 664) eventually fused with the f-PSM. In comparison, 47 % of the docked vesicles fused with the s-PSM while the rest remained docked until the end of the time series.

Fusion of vesicles with the PSM was investigated by analyzing the TR fluorescence over time in a 4x4 pixel² ROI on the center of mass of the vesicle. Figure 6.12 depicts a series of fluorescence micrographs of a vesicle that fuses with the f-PSM and the corresponding TR fluorescence intensity. The time point of fusion was set to t = 0 s.

Due to the high mobility, the vesicles had to be tracked during the analysis inside the f-PSM. Docking of the vesicle in the ROI leads to an increase in TR intensity to a constant level (t = -4.7 s). The onset of fusion (t = 0 s) is indicated by a sudden decrease in TR intensity to the pre-docking intensity when the lipid material radially distributes in the surrounding planar membrane. The time between the initial docking and onset of fusion is called docking time (t_dock) and is analyzed in detail in Chapter 6.4.7.

The radial TR distribution around the fusing vesicles is a certain indicator for successful lipid mixing between the vesicle membrane and the PSM and can be observed in a rectangular donut shaped ROI placed around the vesicle as shown in Figure 6.13A.

The donut-shaped ROI (2) has a size of 8x8 pixel² with a width of 2 pixel while the enclosed center ROI (1) has a size of 4x4 pixel². Time resolved TR fluorescence intensity traces observed in the donut-shaped ROI and in the enclosed center ROI are depicted in Figure 6.13B. Upon onset of fusion (t= 0 s), the TR intensity in the center ROI (black) decays to the pre-docking intensity. At the same time, TR diffuses from the vesicle into the donut-shaped ROI (gray), resulting in an increase of TR intensity, which then immediately decreases again as the TR distributes further in the planar membrane. It was analyzed, whether the TR decay in the center ROI resembles a purely diffusive character by interpreting the vesicle as a lipid disc source that diffuses

Figure 6.12. Fluorescence micrograph sequence of a 240 nm syb 2 vesicle composed of DOPC/POPE/POPS/cholesterol (5:2:1:2) labeled with 1 mol % TR fusing with a planar

∆N-complex containing f-PSM and the corresponding TR fluorescence intensity trace recorded in a 2x2 pixel² ROI on top of the vesicle. The PSM is composed of the same lipid mixture as the vesicle and labeled with 1 % Atto488. Scale bar: 1µm.

into the surrounding PSM with a given diffusion coefficient D. The diffusion of a radial disc source with radius r diffusing into the surrounding membrane according to Fick’s second law can be described by:

I(t) = 1−exp − r² 4Dt

!

(6.3) with I(t) the TR intensity as a function of time,r the radius of the disc source and D the diffusion coefficient [109]. The TR decay shown in Figure 6.13B was modeled with Equation 6.3 assuming a ROI radius with r = 0.5µm and a diffusion coefficient of D = 2µm² s⁻¹ (red curve). The decay can be modeled very well proving a diffusive distribution of TR in the surrounding membrane upon lipid mixing. The assumed diffusion coefficients of 2µm² s⁻¹ does not exactly resemble the diffusion coefficients of the f-PSM as the exact radius of the fusing vesicle is unknown and the fluorophores diffuse through the fusion stalk rather than the PSM.

Fusion of 90 and 240 nm vesicles with the s-PSM was analyzed according to vesicles that fused with the f-PSM by reading out the TR intensity over time (Fig. 6.15).

Docking of the vesicle is observable by the increase in TR fluorescence while the onset of fusion is indicated by the TR decay to the pre-docking intensity as shown for the

Figure 6.13. Analysis of lipid mixing on the f-PSM. (A) Fluorescence micrograph depicting a 240 nm syb 2 vesicle docked to the f-PSM prior to fusion. Scale bar: 1µm.

(B) Time resolved TR fluorescence intensity traces recorded from the center ROI (1, black curve) and from the donut-shaped ROI (2, grey curve) shown in (A). The TR decay recorded in the center ROI was modeled with equation 6.3 assuming a radius ofr

= 0.5µm and a diffusion coefficient of D= 2µm² s⁻¹ (red curve).

events on the f-PSM. In contrast to vesicles that fused with the f-PSM, here, the radial TR distribution could not be observed in a donut-shaped ROI placed around the vesicle due to the gold layer that quenches the fluorescence on the planar membrane (Fig.

6.15A/B). The gold-induced fluorescence enhancement and quenching additionally affects the kinetics of the TR decay in the center ROI, which is why the TR decay cannot be adequately modeled on the s-PSM with equation 6.3 assuming the same parameters (r = 0.5µm and D = 2µm² s⁻¹) as successfully applied on the f-PSM (Fig. 6.15).

Figure 6.14. Fluorescence micrograph sequence of a 240 nm syb 2 vesicle composed of DOPC/POPE/POPS/cholesterol (5:2:1:2) labeled with 1 mol % TR fusing with a planar

∆N-complex containing s-PSM and the corresponding TR fluorescence intensity trace recorded in a 2x2 pixel² ROI on top of the vesicle. The PSM is composed of the same lipid mixture as the vesicle and labeled with 1 % Atto488. Scale bar: 1µm.

Figure 6.15. Analysis of lipid mixing on the s-PSM. (A) Fluorescence micrograph depicting a syb 2 vesicle docked to the s-PSM prior to fusion. Scale bar: 1µm. (B) Time resolved TR fluorescence intensity traces recorded from the center ROI (1, black curve) and from the donut-shaped ROI (2, grey curve) shown in (A). The TR decay recorded from the center ROI was modeled with equation 6.3 assuming a radius ofr = 0.5µm and a diffusion coefficient ofD = 2µm² s⁻¹ (red curve).