Lateral membrane tension of micrometer-sized protruded

microscopy

The distribution of the tension determined for micrometer-sized pore-spanning membranes was too large for calculating membrane tensions with sufficient accu-racy using the fluorescence based approach applying the Young-Laplace theory. To directly measure the lateral tension of protruded pore-spanning membranes atomic force microscopy experiments were conducted. They allow to probe the response of protruded pore-spanning membranes by indentation with a cantilever in order to determine the membrane tension with high accuracy. For fluorescence microscopy

experiments porous silicon substrates were used and the PSMs were imaged using an upright microscope. Spreading of GUVs led to the formation of membrane patches which were localized by fluorescence microscopy for the AFM experiments.

Localization using an upright microscope was not feasible in combination with atomic force microscopy measurements and therefore an inverse microscope had to be used requiring transparent substrates. Pore-spanning membranes were formed on porous glass substrates with a porosity of 20 %, pore radii of 1.75 µm and a pore height of 8 µm. Unfortunately, a fraction of the pores was interconnected as an artifact of the manufacturing process (Figure 4.18). Interconnected pores were not sealed after formation of a pore-spanning membrane as water could be exchanged with the bulk solution via the connection. This led to a breakdown of the osmolarity gradient between pore interior and bulk solution. Therefore, membranes formed on interconnected pores were not protruded by the applied osmolarity gradient and remained planar. As a result of the interconnection protruded and planar pore-spanning membranes were probed after application of an osmolarity gradient on the same substrate.

Figure 4.18:Representative scanning electron microscopy image of the cross section of a porous SiO₂substrate with pore radii of 1.75 µm, a pore depth of 8 µm and a porosity of 20 %. The pores are partially interconnected at the pore bottom (red circles). Scale bar:

5 µm.

A typical force volume image of pore-spanning membranes measured on porous SiO₂ substrates after application of an osmolarity gradient of 20 mOsmol/L is shown in Figure 4.19 A. Each pixel represents a force-distance curve of the cantilever approaching the sample and indenting it until a previously defined force is reached. The color code indicates the absolute height when the predefined force

was reached and the indentation stopped. Representative force-distance curves measured on non-covered pores, covered open pores and membrane-covered closed pores are shown in Figure 4.19 B.

The assignment of a pore being closed or open was achieved by measuring the membrane height. The height of the pore-spanning membrane was calculated by subtracting the absolute height of the contact point of the cantilever with the pore rim (Figure 4.19 B, black) from the contact point of the cantilever with the pore-spanning membrane (Figure 4.19 B, blue, green, red). Non-covered pores adjacent to protruded membranes led to a decrease of the membrane height of the protruded PSMs. If the membrane height was larger than 100 nm after applying an osmolarity gradient, a pore was assumed to be closed since the membrane was protruded by the applied osmolarity gradient (Figure 4.19 A, blue). Membranes with a height of

−100 nm to 100 nm were considered to span an open pore (Figure 4.19 A, green).

For non-covered pores apparent heights smaller than −100 nm were measured (Figure 4.19 A, red).

Figure 4.19: A: Force volume image of pore-spanning membranes (DPhPC/Texas Red, 99:1) after application of an osmolarity gradient of 20 mOsmol/L. Protruded (blue circle) and planar (green circle) pore-spanning membranes were imaged together with non-covered pores (red circle). Pore diameter: 3.5 µm.B: Representative force-distance curves measured in the center of the regions marked inAand on the pore rim (black curve). The contact points are marked with diamonds.

Fluorescence microscopy of protruded pore-spanning membranes revealed mem-branes located at the border of a membrane patch having smaller heights compared to membranes in the patch center (Figure 4.15). Using atomic force microscopy, it could be shown that the height of membranes neighbored to an open pore was

reduced similar to membranes located at the border of the membrane patch as observed by fluorescence microscopy. Due to the large number of open pores a correlation of the height of the protruded pore-spanning membranes measured by atomic force microscopy to the applied osmolarity gradient was not possible.

The overall mechanical response to indentation is given by the slope of the force-distance curves. It is mainly determined by pre-stress of the membrane caused by adhesion to the substrate.^{[119, 141]} The lateral membrane tension was calculated for the experimentally used parameters such as the radius of the cantilever tip (r_tip= 25 nm), the pore radius (r_pore= 1.75 µm) and the apparent spring constant.

Latter was obtained by linear fitting of the part of the force-distance curves where the cantilever indented the membranes (Figure 4.19 B, magenta).^[120] Lateral membrane tensions of 2±1 mN/m (n= 171) measured for protruded pore-spanning membranes on closed pores and 2±1 mN/m (n= 94) measured on open pores were determined. Comparison of the membrane tensions measured with atomic force microscopy with those determined by fitting the height of protruded pore-spanning membranes measured with confocal fluorescence microscopy shows that all membrane tensions are within the expected range of 1-3 mN/m measured for PSMs of different lipid compositions on hydrophilically functionalized porous substrates with open pores.^{[119, 139]}

The lateral tension of lipid bilayers was successfully measured by optical micros-copy using protruded pore-spanning membranes on porous substrates with pore radii of 425 nm. For larger pores with radii of 1.75 µm the distribution of the membrane tensions was too broad for an accurate determination of the membrane’s lateral tension. It could be shown that the model system of protruded pore-spanning membranes can be used to analyze membrane-protein interactions involv-ing changes in the lateral membrane tension. Bindinvolv-ing of ENTH to protruded pore-spanning membranes leads to growth and deflation of pore-pore-spanning membranes.

Uncertainties of the osmolarity measurements and the large influence of the pore geometry resulted in a high deviation of the individual measurements limiting the application. To overcome these experimental limitations and to perform experiments with lipid bilayers having a lower membrane tension, experiments with giant unilamellar vesicles were conducted.

4.5 Adhered GUVs as a model system to analyze