Single-vesicle fusion assay

To investigate the kinetics and fusion pathways of SNARE-mediated membrane fusion, single-vesicle content release experiments of LUVs fusing with PSMs were recorded by means of SDCM.

Experimental procedure

For two channel experiments, SRB filled LUVs containing syb 2 and N49 doped GUVs lipid labeled with 1 mol% Atto655 DPPE were prepared according to Chapter 3.2.4 and GUVs were spread on porous substrates with dpore = 1.2 m according to Chapter 3.2.5. Using an upright spinning disc setup (Yokogawa CSU-X, Rota Yokogawa KG, Wehr, Germany) SRB was excited at ex = 561 nm and Atto655 DPPE at ex = 639 nm. The emitted light was focused on either part of the EMCCD camera (iXon 897 Ultra, Andor technology, Belfast, United Kingdom, pixel size 222 × 222 nm²) using an optosplit II. The optosplit was equipped with a H 643 LPXR superflat beamsplitter, a 595/40 ET bandpass, and a 655 LP ET longpass emission filter (AHF Analysetechnik AG, Tübingen, Germany) with properties shown in Figure 3.25.

After laser alignment the micro injection unit for LUV addition was focused near the focal point of the objective (LUMFLN 60xW, NA 1.1, Olympus, Hamburg, Germany) and filled with LUV

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solution diluted by a factor of 10 with SNARE buffer B. One PSM was focused and 1 L of LUV solution added directly on top of the PSM. This allowed for uniform vesicle addition between different measurements. Docking to and fusion of LUVs with the PSM was recorded with microscopy settings listed in Table 3-13.

Table 3-13 Microscopy settings to monitor single-vesicle fusion experiments on PSMs in dual color mode.

User settings laser power 561 22 %

laser power 639 10 % exposure time 0.02 s electron multiplier gain 800

framerate 48 fps or 20.83 ms per frame

N (frames) 20000

total time 6.94 min

Figure 3.25 Filterset used for two channel single-vesicle fusion experiments. (A) 595/40 ET bandpass filter, (B) 655 LP ET longpass filter and (C) H 643 LPXR superflat beamsplitter.

Data evaluation

Since SRB fluorescence intensity partially emits into the Atto655 DPPE channel and all single-vesicle fusion experiments were performed under the same conditions, a global factor for crosstalk correction (CF) was determined. Therefore, SRB filled LUVs were immobilized on a functionalized porous substrate and imaged under the same experimental conditions and

51 microscopy settings as for single-vesicle experiments. Using ImageJ, the sum of the time series was calculated, vesicles tagged on either side of the split image (left: l, right: r) and background corrected (BGl, r) maximum fluorescence intensities (Imax) extracted. CF was then calculated according to Equation (3-10).

𝐶𝐹 =1

𝑛∑(𝐼_max,l,𝑖− 𝐵𝐺_l) (𝐼_max,r,𝑖− 𝐵𝐺_r)

𝑛

𝑖=1

(3-10)

Tif-stacks of single-vesicle fusion experiments recorded by means of SDCM were loaded into ImageJ and vesicles that docked mobile to the f-PSM, were tracked using the ImageJ plugin Mosaic.^[103] Immobile vesicles docked to the s-PSM were evaluated using a custom made MATLAB-script illustrated in Figure 3.26 which can be downloaded at https://github.com/imey78/FusionAnalysis.git. Each time series was automatically split by the software of the microscope into up to 6 different Tif-stacks, each stack loaded into ImageJ, the sum created, and docked vesicles tagged with a minimum 4 x 4 pixel ROI. ROI-coordinates as well as Tif-stacks were opened with “program 1” in MATLAB. When measuring in the two channel mode, the different halves of the transmitted image were aligned by marking characteristic points such as pore centers on either side of the split image. The ROIs of tagged vesicles in the SRB channel and an additional ROI for dynamic background correction were then transferred to the membrane channel and fluorescence intensity as a function of time read out from the raw data. The saved fluorescence intensity time traces were opened in “program 2”

and crosstalk corrected fluorescence intensity of each ROI n plotted as a function of time. Each individual event was then assigned to a specific group using a unique tag, event-tags saved, and the process repeated for all recorded time series m. Fluorescence intensity time traces i of the same event-tag were extracted by and opened in “program 3” to set time points of interest e.g.

the time of vesicle docking. This data could then be used for further evaluation of e.g. fusion kinetics.

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Figure 3.26 Process chart illustrating the evaluation of vesicle docking to and fusion with the s-PSM. ImageJ was used to load all Tif-stacks of each time series and manually tag docked vesicles with a ROI. ROI-sets and Tif-stacks were opened with program 1, membrane channels aligned and background ROIs selected. Fluorescence intensity time traces were saved, opened in program 2, and the crosstalk corrected. Specific event-tags were assigned to each docked vesicle n, data saved, and all steps repeated for all recorded time series m. Program 3 scanned the complete data set for identical event-tags and loaded the respective fluorescence intensity time traces. Important time points were set and the data saved for further evaluation.

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4 Results

-Parts of this work have been published in Biophysical Journal^[95]-

Single-vesicle fusion assays mimicking the process of membrane fusion prior to neurotransmitter release in synapses have been developed over the past decades.[10,48,65,75,77,104]

These in vitro assays helped to better understand the molecular mechanisms and kinetics of SNARE-mediated membrane fusion and usually monitored fusion of highly curved vesicles with a planar supported lipid bilayer (SLB) on a single-vesicle level by means of fluorescence microscopy.^[48,75,77] Vesicles were filled with water soluble dyes to directly monitor fusion pore formation, however, SLBs lack of a second aqueous compartment for the content to be released in.[36,74,82,105] A model membrane system that is both planar, thus easily accessible by fluorescence microscopy, and provides enough space on both sides of the membrane are pore-spanning membranes (PSMs). Based on the work of Ines Höfer, Lando Schwenen recently developed a lipid mixing based single-vesicle fusion assay using PSMs that led to valuable insights in the process of SNARE-mediated membrane fusion.^[84,85]

In the present work this model system was extended to directly visualize fusion pore formation by means of content release while simultaneously lipid mixing was monitored to gather detailed information about different fusion pathways. To achieve this, the v-SNARE synaptobrevin 2 was reconstituted into large unilamellar vesicles (LUVs) filled with the water-soluble fluorophore sulforhodamine B (SRB) and the t-SNARE acceptor complex N49 reconstituted into PSMs that were lipid labeled with Atto655 DPPE (Figure 4.1 A). Single-vesicle docking to and fusion with the PSM was then recorded by means of high speed dual color spinning disc confocal microscopy (Figure 4.1 B).

Figure 4.1 (A) Schematic illustration of the single-vesicle content release assay based on PSMs and SRB filled LUVs. (B) Fluorescence micrograph of Atto655 DPPE labeled PSMs (false colored in green) with reconstituted ΔN49-complex (DOPC/POPE/POPS/cholesterol/Atto655 DPPE; 5/1.9/1/2/0.1 (n/n), nominal p/l 1:500).

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Fluorescence intensity is quenched due to the underlying gold surface inside the solid supported part of the PSM (s-PSM). Proteo-LUVs (false colored in magenta) containing syb 2 and filled with SRB (43 mM, same lipid composition and p/l ratio) are docked to the s-PSM. Scale bar: 2 μm.