- FRAP experiments were carried out by Kira Herwig as part of her bachelor thesis -
4.3.1 Mobility of lipids and N49-complexes in GUVs
The mobility of lipids and proteins is important for the process of SNARE-mediated membrane fusion in vivo and therefore it is necessary to investigate the mobility of components in the respective model membrane systems.[108] As GUVs are the starting material to form pore-spanning membranes (PSMs) and known to enable free 2D diffusion of molecules inside their membrane, they are used as a reference system in this work. Thus, the diffusion coefficient of the t-SNARE acceptor complex N49 was first quantified in free GUVs by means of fluorescence recovery after photobleaching (FRAP) experiments. Proteo-GUVs (DOPC/POPE/POPS/cholesterol/Atto390 DPPE; 5/1.95/1/2/0.05 (n/n), nominal p/l 1:250) were prepared and FRAP experiments performed as described in Chapter 3.2.4 and Chapter 3.3.2, respectively. Briefly, fluorescence was bleached in a circular ROI in the top plane of the GUV (Figure 4.5 A, white ROI, rn = 3.17 m) and fluorescence recovery recorded as a function of time. The data was corrected for focus drift/photofading during ilumination and background fluorescence intensity. The specific diffusion time 1/2 was extracted by fitting Equation (3-7) to the normalized, corrected recovery curve (Figure 4.5 B, red line).[97] To compensate for diffusing molecules into the ROI during bleaching, the effective bleach radius re was extracted from the postbleach profile (Figure 3.21 B, C). The diffusion coefficient D was then calculated using Equation (3-6) and the mobile fraction Fm of molecules using Equation (3-8).[98]
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Figure 4.5 (A) Fluorescence micrographs of a FRAP experiment on the top plane of a GUV (DOPC/POPE/POPS/cholesterol/Atto390 DPPE; 5/1.95/1/2/0.05 (n/n), nominal p/l 1:250). After acquisition of pre bleach fluorescence (I) lipid dye molecules were bleached (white circle, rn = 3.17 m, re = 6.67 m) and fluorescence recovery was monitored as a function of time. Scale bar = 10 m. (B) Normalized fluorescence recovery as a function of time was read out from the white ROI in A. The red line is the fit of Equation (3-7) to the data leading to the diffusion time 1/2 =0.52 s.
Both Atto390 DPPE and N49 were fully mobile inside the GUV membrane with Fm (Atto488 DPPE) = 100 ± 8 % and Fm (N49-Atto488) = 100 ± 7 %. Fitting a normal distribution function to the histogram of diffusion coefficients (Figure 4.6 A, B, red line) results in diffusion coefficients of DAtto390 DPPE = 9 ± 3 m2 s-1 (N = 25) and DN49-Atto488 = 5 ± 3 m2 s-1 (N = 26) with standard deviations as errors.
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Figure 4.6 Diffusion coefficients of lipids and N49-Atto488 inside GUVs. The red lines are the result of fitting a normal distribution to the data with median values for the diffusion coefficient of lipids DAtto390 DPPE = 9 ± 3 m2 s-1 (N = 25) (A) and proteins DN49-Atto488 = 5 ± 3 m2 s-1 (N = 26).
4.3.2 Mobility of lipids and N49-complexes in PSMs
Lipid and t-SNARE mobility in pore-spanning membranes (PSMs) is essential for the fusion of v-SNARE doped vesicles with the target membrane. PSMs were prepared as described in Chapter 3.2.5 by spreading GUVs with reconstituted N49-Atto488 complex (DOPC/POPE/POPS/cholesterol/Atto390 DPPE; 5/1.95/1/2/0.05 (n/n), nominal p/l 1:250) on porous substrates with pore diameters of 5 m. PSMs consist of two distinct membrane parts, a freestanding part spanning the holes of the substrate (f-PSM) and a solid supported part covering the gold/mercapto-hexanol functionalized pore rim (s-PSM). As molecules exhibit different diffusive behavior in the two different parts of the PSM they have to be investigated separately. Previously, Schwenen et al. performed fluorescence correlation spectroscopy (FCS) experiment inside the f-PSM using a labeled transmembrane domain (TMD) of syx 1A and labeled syx 1A (aa 183-288) as well as lipid markers.[85] The obtained diffusion coefficients were Dsyx 1A-TMD (f-PSM) = 3.4 ± 0.2 m2 s-1, Dsyx 1A (f-PSM) = 2.3 ± 0.5 m2 s-1, and DDPPE (f-PSM) = 7.7 ± 0.4 m2 s-1. Due to the quenched fluorescence inside the s-PSM, neither FCS experiments nor FRAP experiments as described for GUVs could be performed in this part of the PSM. However, by bleaching the fluorescence of one individual f-PSM the recovery of f-PSM fluorescence is dominated by the diffusion of molecules over the s-PSM surrounding the pore. These so called indirect FRAP experiments, as previously reported by Kuhlmann et al. for the syx-TMD, can be used to describe the diffusion of molecules inside the s-PSM.[61]
Conventional data analysis where the diffusion coefficient is directly derived from the half-life time of fluorescence recovery, as used in Chapter 4.3.1, is not applicable for indirect FRAP
63 experiments due to two reasons. First, the effective bleach radius could not be extracted from the postbleach profile since the Gaussian intensity profile is cut off at the edge of the pore, due to the lack of fluorescence intensity on the pore rim. Secondly, the recovery of fluorescence inside the bleach ROI is influenced by the diffusion of molecules over the pore rim but also inside the f-PSM. Such a two component system would need a higher signal to noise ratio of the data than achieved in this work to lead to accurate values of 1/2 extracted from the recovery curve.[100] Thus a method was used which is based on the comparison of recovery curves with simulated FRAP experiments.[61] Briefly, FRAP experiments were performed as described in Chapter 3.3.2 with exemplarily fluorescence micrographs shown in Figure 4.7 A. Finite element simulations were performed as described in Chapter 3.3.2 to model the 2D diffusion of lipids and proteins inside the PSM after bleaching of fluorescence of a f-PSM (Figure 4.7 B). Values for the diffusion inside the f-PSM were fixed to DLipid (f-PSM) = 7.7 μm2 s-1 and DProtein (f-PSM) = 3.4 μm2 s-1 (values derived from FCS measurements) and fluorescence recovery inside the bleach ROI read out as a function of time.[85] Within the observation time lipids showed full mobility and proteins a mobile fraction of 70 ± 21 %. These values are not quantitative mobile fractions, as only the mobile parts of molecules are imaged in this setup.
The mean, normalized, corrected time resolved fluorescence recovery from indirect FRAP experiments was then plotted against recovery curves obtained from finite element simulations derived using different diffusion coefficients for the s-PSM (Figure 4.7 C, D). Comparing simulated and experimental recovery curves lead to the estimated diffusion coefficients of DDPPE (s-PSM) = 2 ± 1 m2 s-1 and DΔN49 (s-PSM) = 1.0 ± 0.5 m2 s-1.[95]
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Figure 4.7 Indirect FRAP experiments and finite element simulations to determine the diffusion coefficient of lipids and proteins inside the s-PSM (DOPC/POPE/POPS/cholesterol/Atto390 DPPE; 5/1.95/1/2/0.05 (n/n), nominal p/l 1:250) (A) Fluorescence micrographs of an indirect FRAP experiment on PSMs (dpore = 5 m).
N49-Atto488 fluorescence is bleached inside the white ROI (r = 2.2 m) and fluorescence intensity was monitored over time. Scale bars = 10 m. (B) Snapshots of a simulated indirect FRAP experiment obtained from finite element simulations. Fluorescence intensity is bleached inside the red ROI (r = 2.2 m) and fluorescence recovery was monitored over time. (C) Averaged, corrected, normalized fluorescence recovery curve obtained from bleaching Atto390 DPPE labeled f-PSMs (N = 33, open squares). Simulated recovery curves with DDPPE (s-PSM) = 2 (black line), 3 (red line), and 4 (blue line) μm2 s-1 with fixed DLipid (f-PSM) = 7.7 μm2 s-1. (D) Averaged, corrected, normalized fluorescence recovery curve obtained from bleaching N49-Atto488 containing f-PSMs (N = 33, open squares). Simulated recovery curves with DN49 (s-PSM) = 0.5 (black), 1 (red), and 1.5 (blue) µm2 s
-1 and DProtein (f-PSM) = 3.4 µm2 s-1.
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