Modulating the Lateral Tension of Solvent-Free Pore-Spanning

Article pubs.acs.org/Langmuir

Modulating the Lateral Tension of Solvent-Free Pore-Spanning Membranes Jan W. Kuhlmann, Ingo P. Mey, and Claudia Steinem* Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstraße 2, 37077 Göttingen, Germany S Supporting Information *

ABSTRACT: The plasma membrane of animal cells is attached to the cytoskeleton, which significantly contributes to the lateral tension of the membrane. Lateral membrane tension has been shown to be an important physical regulator of cellular processes such as cell motility and morphology as well as exo- and endocytosis. Here, we report on lipid bilayers spanning highly ordered pore arrays, where we can control the lateral membrane tension by chemically varying the surface functionalization of the porous substrate. Surface functionalization was achieved by a gold coating on top of the pore rims of the hexagonal array of pores in silicon nitride substrates with pore radii of 600 nm followed by subsequent incubation with various n-propanolic mixtures of 6-mercapto-1-hexanol (6MH) and O-cholesteryl N-(8′-mercapto-3′,6′-dioxaoctyl)carbamate (CPEO3). Pore-spanning membranes composed of 1,2-diphytanoyl-snglycero-3-phosphocholine were prepared by spreading giant unilamellar vesicles on these functionalized porous silicon nitride substrates. Different mixtures of 6MH and CPEO3 provided self-assembled monolayers (SAMs) with different compositions as analyzed by contact angle and PM-IRRAS measurements. Site specific force-indentation experiments on the pore-spanning membranes attached to the different SAMs revealed a clear dependence of the amount of CPEO3 in the monolayer on the lateral membrane tension. While bilayers on pure 6MH monolayers show an average lateral membrane tension of 1.4 mN m−1, a mixed monolayer of CPEO3 and 6MH obtained from a solution with 9.1 mol % CPEO3 exhibits a lateral tension of 5.0 mN m−1. From contact angle and PM-IRRAS results, the mole fraction of CPEO3 in solution can be roughly translated into a CPEO3 surface concentration of 40 mol %. Our results clearly demonstrate that the free energy difference between the supported and freestanding part of the membrane depends on the chemical composition of the SAM, which controls the lateral membrane tension.

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INTRODUCTION The plasma membrane is generally described by the fluid mosaic model comprising a two-dimensional fluid lipid bilayer with embedded proteins.1 Based on this picture, different model membrane systems2−4 have been developed resembling the bilayer structure, the semipermeable barrier function, the lateral mobility of the lipids, and certain functionalities provided by reconstituted membrane proteins. However, the plasma membrane of animal cells is also attached to the cytoskeleton contributing significantly to the mechanical properties of the membrane. It has been shown that the force needed to stretch the membrane of a vesicle, which reflects the in-plane membrane tension, is much smaller than the force needed to deform the plasma membrane as a result of contributions from membrane proteins and membrane-to-cortex attachments (cortical tension).5−7 This overall membrane tension, being the sum of the in-plane membrane tension and the cortical tension, has been shown to be an important physical regulator of cell motility and morphology.8−10 Plasma membrane tension regulates biological processes such as exocytosis (stimulated by high membrane tension) and endocytosis (stimulated by low membrane tension).10,11 Hence, it is highly desirable to © 2014 American Chemical Society

establish model membrane systems that include lateral membrane tension and allows one to control it. Previously, we have shown that planar membranes prepared on a highly ordered array of pores, termed pore-spanning membranes, allowed us to probe their lateral tension by means of indentation experiments performed by the atomic force microscope.12 In the early experiments, pore-spanning lipid bilayers prepared on micrometer-sized pores have been prepared by painting lipids dissolved in an organic solvent onto silicon nitride pore arrays, where the top part of the interpore surface was functionalized with a hydrophobic selfassembled monolayer (SAM) resulting in hybrid membranes.13 These hybrid pore-spanning bilayers showed nonphysiological lateral tension values in the range of 20 mN m−1 due to the large gain in adhesion energy on the pore rim, which prestresses the bilayer close to rupture.14 To be able to generate solvent-free and continuous porespanning lipid bilayers, Kocun et al.15 presented a method Received: May 16, 2014 Revised: June 20, 2014 Published: June 20, 2014 8186

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Fluorescence Microscopy. An upright optical microscope (Olympus BX-51, Olympus Germany GmbH, Hamburg, Germany) equipped with a mirror unit (Olympus, U-MNG2, excitation 530−550 nm, emission >590 nm, dichroic mirror 570 nm) and a water immersion objective (Olympus, LUMPlanFl 40XW, N.A. = 0.8) with 40× magnification was used for fluorescence imaging of the porespanning membranes. The position of each membrane patch on the substrate was identified for the AFM experiments. Atomic Force Microscopy (AFM). Experiments were performed on a MFP-3D AFM (Asylum Research, Santa Barbara, CA) equipped with a top view camera to localize the membrane. MLCT-Au cantilevers (Bruker AFM Probes) with a nominal spring constant of 10 mN m−1 were used for force spectroscopy experiments. The exact spring constant of the cantilever was determined prior to each experiment with the thermal noise method integrated in the Asylum Research software based on IGOR Pro (Wavemetrics). Force volume images with a size of 6 × 6 μm2 were recorded with a resolution of 32 × 32 pixels2 with a load force of 400 pN and a velocity of 3 μm s−1. For the statistical analysis of the apparent spring constants kapp, from which the lateral membrane tension can be extracted, at least three independent membrane preparations were used for each SAM composition. For each SAM composition, 216−735 force−indentation curves were evaluated by picking four curves from the center of each pore-spanning membrane. Polarization Modulation-Infrared Reflection Absorption Spectroscopy (PM-IRRAS). PM-IRRAS samples were mounted in the external beam of a Fourier transform-infrared (FT-IR) spectrometer (Vertex 70, Bruker) equipped with a grid polarizer and a ZnSe photoelastic modulator (PM90, HINDS Instruments, Hillsboro, OR). All measurements were performed at an optimal angle of incidence of 83°. The reflected IR beam was focused on a liquid nitrogen cooled mercury cadmium telluride (MCT) detector. For each run, 367 spectra were collected with a resolution of 4 cm−1. Contact Angle Measurements. Contact angles were determined on a self-built goniometer setup. 5 μL of ultrapure water was deposited on top of the samples and imaged with a camera (Nikon, D600) equipped with a AF-S Nikkor 18/300 mm objective (Nikon) fronted by a custom-made lens (Scientific Precision Instruments GmbH, Oppenheim, Germany). The Young−Laplace equation was fit to the contour of the water droplets to determine the contact angle.19

based on spreading giant unilamellar vesicles (GUVs) onto OH-terminated SAMs on a gold-functionalized pore array in silicon nitride. The bilayers exhibit a one order of magnitude lower membrane tension of about 1−3 mN m−1. These results show that the prestress of pore-spanning membranes is a function of the free energy difference between the supported and free-standing membrane and can thus be controlled by the surface functionalization.12,13 To analyze the influence of the functionalization of the pore rims on the lateral prestress of the pore-spanning membranes in more detail, we set out experiments where we varied the composition of the SAMs. Besides using 6-mercapto-1-hexanol (6MH) to generate OHterminated SAMs, we increased the hydrophobic character of the SAMs by adding O-cholesteryl N-(8′-mercapto-3′,6′dioxaoctyl)carbamate (CPEO3), which we have previously shown to allow spreading of GUVs to obtain pore-spanning membranes.16 The amount of CPEO3 in the SAM greatly increased the SAM’s hydrophobicity resulting in an increased tension of the pore-spanning membranes, verifying our hypothesis that the large adhesion energy on the pore rims is the main source of membrane tension.

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MATERIALS AND METHODS

Materials. 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was purchased from Avanti Polar Lipids (Alabaster, AL). Sulforhodamine-1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (Texas Red DHPE) and 6-mercapto-1-hexanol (6MH) were from Sigma-Aldrich. O-Cholesteryl N-(8′-mercapto-3′,6′-dioxaoctyl)carbamate (CPEO3) was synthesized as described previously.17 Porous silicon nitride substrates with pore radii of 600 nm were purchased from fluXXion B.V. (Eindhoven, NL). Preparation of Substrates. Porous silicon nitride substrates were rinsed with ethanol p.a. and dried under a stream of nitrogen. The surface was first sputter-coated (Cressington 108auto, Cressington Scientific Instruments Ltd., Watford, UK) with titanium (10 s, 40 mA, 0.7 mbar). Then, a 30−40 nm thick gold layer was evaporated on top of the titanium layer at a deposition rate of 0.3 nm s−1 (Bal-Tec Med 020, Balzer, Liechtenstein). The gold-coated substrates were subsequently immersed in 1 mM n-propanolic thiol solutions of 6MH and CPEO3 mixtures and incubated overnight at room temperature. Solid substrates for contact angle and polarization modulationinfrared reflection absorption spectroscopy (PM-IRRAS) measurements were obtained by coating microscope glass slides with a 3 nm thick chromium and a 200 nm thick gold layer. The substrates were cleaned as described for the porous silicon nitride substrates. SAM formation was carried out in 1 mM n-propanolic thiol mixtures of 6MH and CPEO3 at room temperature overnight. After monolayer formation, the samples were rinsed with ethanol p.a. and dried under a stream of nitrogen. Vesicle Preparation. Giant unilamellar vesicles (GUVs) were prepared employing electroformation.18 DPhPC dissolved in chloroform (1.7 mg mL−1) was deposited on two indium tin oxide (ITO) coated glass slides and dried in air. Remaining chloroform was removed under vacuum (10 min, 1 mbar). A Teflon ring was placed between the two ITO slides (the lipid films orientated face to face), and the enclosed volume was filled with sucrose solution (0.2 M). A frequency generator was connected to the ITO slides, and a sine wave was applied for 3 h at 400 V m−1 and 10 Hz to receive GUVs. Preparation of Pore-Spanning Membranes. SAM coated porous substrates were rinsed with ethanol p.a., dried under a stream of nitrogen, and placed in a buffer-filled (20 mM HEPES, 100 mM KCl, pH 7.4) home built measuring chamber. Sucrose-filled GUVs were sedimented in buffer to dilute the surrounding sucrose solution. 5 μL of the sedimented GUV suspension was pipetted on top of the porous substrate for spontaneous GUV rupture and formation of solvent-free pore-spanning membranes.

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RESULTS AND DISCUSSION Formation of Pore-Spanning Membranes. To prepare pore-spanning lipid bilayers, microstructured silicon nitride substrates with hexagonal arrays of pores with radii of 600 nm were used.20 The top part of the substrates was coated with a 30−40 nm thick gold layer to allow for the chemisorption of thiol monolayers (Figure 1E). The scanning electron micrograph (Figure 1A) reveals that the top part of the porous substrate is homogeneously covered with gold, while the pore walls are almost completely gold-free, ensuring that a dense SAM is only formed atop the porous substrate. By changing the mole fraction of CPEO3 χCPEO3 in the binary mixture in solution, the amount of CPEO3 adsorbed onto the gold surface can be varied.21 After monolayer formation and rinsing with npropanol and buffer, DPhPC GUVs labeled with Texas Red DHPE were spread onto the functionalized substrates to generate pore-spanning membranes. The maximal applicable lateral membrane tension is limited by the area compressibility modulus of the membrane. DPhPC has an area compressibility modulus of Ka = 670 mN/m, whereas typical fluid phase lipids have area compressibility moduli of only around 200−250 mN/m.22 Even though we have shown that pore-spanning membranes composed of fluid phase lipids like DOPC and POPC can be readily obtained and indented (Figure S1, Supporting Information),15,23 they are more prone to rupture during an indentation experiment, and 8187

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pore-spanning membrane patches were located on the substrate by fluorescence microsopy and then analyzed by means of force volume experiments. For each membrane preparation, force volume AFM images were obtained (Figure S2, Supporting Information). Within a force map, force−indentation curves on the pore rims, on pore-spanning membranes, and on uncovered pores are clearly distinguishable from each other. Figure 2A

Figure 1. (A) Scanning electron micrograph of a gold-coated silicon nitride substrate with a hexagonal pore array. The surface has a porosity of 30%, and the pores display a radius of 600 nm. Scale bar: 1 μm. (B−D) Fluorescence images of pore-spanning DPhPC membranes labeled with Texas Red DHPE recorded 30 min after GUV spreading on differently functionalized pore rims. Mixed SAMs of 6MH and CPEO3 with (B) χCPEO3(sol) = 4.8 mol %, (C) χCPEO3(sol) = 7.1 mol %, and (D) χCPEO3(sol) = 9.1 mol %. Scale bar: 20 μm. (E) Schematic drawing of a pore-spanning membrane attached to a mixed monolayer composed of 6-mercapto-1-hexanol and the cholesterol derivative CPEO3.

Figure 2. (A) Force−indentation curves observed on a pore rim and on pore-spanning membranes on SAMs composed of 6MH and CPEO3. From the linear force response upon indentation the apparent spring constant kapp of the system can be extracted. (B) Box plot diagram showing the lateral membrane tension obtained from the force−indentation experiments as a function of the mole fraction of CPEO3 in the SAM solution. The ends of the whiskers mark the minimum and maximum values and the black diamonds the mean values.

thus we used DPhPC in this study to prevent artifacts arising from membrane rupturing to clearly demonstrate how surface chemistry influences lipid bilayer mechanics. Figures 1B−D show fluorescence images of pore-spanning membrane patches on three different SAMs obtained from thiol solutions composed of 6MH and CPEO3 containing 4.8, 7.1, and 9.1 mol % of CPEO3. Images were taken 30 min after the GUV spread on the surface. The red fluorescence intensity indicates an intact bilayer spanning the pores.15 The pore rims appear darker due to quenching of the fluorescence by the underlying gold layer.24−26 While the formed pore-spanning membranes on OH-terminated SAMs are stable for hours independent of whether mercaptoethanol15,23 or mercaptohexanol (this study) is used for SAM formation, the stability decreases with the amount of CPEO3 in the binary mixture indicated by individually ruptured pore-spanning membranes within the membrane patch (Figure 1C, D).14 The topology of the pore-spanning membranes is rather planar, giving the dimensions of the pores of 1.2 μm. We expect the membrane to be slightly bent as depicted in Figure 1E to follow the gold-covered surface,27 and in our AFM experiments, we observed that the suspended membrane can be located a few ten nanometers below the surface of the porous support. Mechanical Properties of Pore-Spanning Membranes. To gather information about the lateral tension of the membranes on the differently functionalized pore rims, the

shows typical force−indentation curves obtained in the center of a pore-spanning membrane each prepared on a SAM with a different CPEO3 content. The force−indentation curve monitored on the pore rim serves as a reference for the position of the contact point on the pore-spanning membrane. All curves show a linear response, lack hysteresis, and the slope of the response is independent of the loading rate, as previously reported for force−indentation curves on pore-spanning membranes on hydrophobically and hydrophilically functionalized substrates.13,15,28 The maximum indentation depth we found in our experiments was up to 300 nm for membranes on CPEO3-containing SAMs and more than 500 nm for membranes on pure 6MH SAMs. We rarely observed membrane rupturing during indentation experiments. Such indentation depths translate into an area increase of more than 10%, indicating that even for membranes on CPEO3containing SAMs a lipid reservoir is still available. If no lipid reservoir is accessible upon indentation, as is the case for membranes on hydrophobic and densely packed alkanethiol 8188

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SAMs, membrane rupture occurs already at an area dilatation of roughly 3%.13 The linear slope we have termed apparent spring constant kapp, which represents the overall mechanical response of the membrane upon indentation.13 kapp is dominated by the lateral membrane tension (e.g., prestress) as a result of the free energy difference between the supported membrane on the pore rim and the free-standing membrane, which spans the pore as previously discussed.13,28,29 Excluding stretching and bending of the membrane and assuming an infinite lipid reservoir on the pore rim, the lateral membrane tension σ can be obtained from eq 1: h=

⎡ ⎛ FR ⎞⎤ F ⎢ tip ⎟⎥ 1 − ln⎜⎜ 2⎟ 4πσ ⎢⎣ ⎝ 2πσR pore ⎠⎥⎦

(1)

where h is the indentation depth, F the force, Rtip the indenter radius, and Rpore the pore radius.29 Equation 1 was solved using Mathematica 6.0 (Wolfram Software) for indentation depths of 30 nm using an indenter radius of 20 nm and a pore radius of 600 nm. Several hundred force−indentation curves were evaluated to determine the lateral membrane tension σ (Figure 2B). It becomes obvious that σ increases from 1.4 mN m−1 for pore-spanning membranes on pure 6MH to 5.0 mN m−1 if a SAM was formed from a solution containing 9.1 mol % of CPEO3. These findings provide strong evidence that the adhesion, i.e. the hydrophobicity of the SAM, between the lipid leaflet facing the support and the SAM strongly influences the lateral tension in the free-standing membrane. However, while the ratio between 6MH and CPEO3 in solution is known and can be readily controlled, the actual ratio in the SAM can differ considerably.30−32 This is due to the fact that each component in a binary thiol mixture has different binding kinetics and binding energies,33 resulting in a composition of a SAM that deviates from the solution composition. Hence, to be able to relate the amount of CPEO3 in the SAM to the observed lateral membrane tension, we analyzed the hydrophobicity of the SAM by contact angle measurements and estimated the chemical composition of the SAMs by PM-IRRAS experiments. Contact Angle Measurements. While 6MH exposes a hydrophilic OH group to the aqueous phase capable of forming hydrogen bonds, the larger and bulkier cholesterol derivative CPEO3 is much more hydrophobic. By changing the ratio of the thiols in solution, it is expected that the hydrophobicity of a gold-covered surface increases with increasing amount of CPEO3 in solution.33 To analyze the hydrophobicity of the SAMs on the gold surfaces, contact angle measurements were performed (Figure 3). As expected, the contact angle increases from 20° to 110° with increasing mole fraction of CPEO3 in solution (Figure 3A). Between 0 and 15 mol %, the contact angle increases in a slightly S-shaped manner possibly as a result of the complex binding kinetics of binary SAMs including multiple 2D-phase transitions, island nucleation, and growth.33 Of note, saturation of the contact angle occurs already at 15 mol % CPEO3 in solution. Even if the CPEO3 mole fraction is further increased in solution, the contact angle remains constant, indicating that the surface has reached its maximum occupancy of CPEO3. This result is in close agreement with findings of Jeuken et al.,31 where mixed SAMs composed of CPEO3 and 6MH were studied by electrical impedance spectroscopy and X-ray photoelectron spectroscopy. They found that at a CPEO3

Figure 3. (A) Contact angles of sessile water droplets on SAMs composed of different ratios of 6MH and CPEO3. The data depicted as hollow diamonds are those values that correspond to the mixtures used for the generation of pore-spanning membranes. (B) Contours of water droplets on SAMs of 6MH and CPEO3 on gold coated glass slides with different mole fractions of CPEO3 (1) 0 mol %, (2) 4.8 mol %, (3) 6.3 mol %, (4) 7.7 mol %, (5) 9.1 mol %, and (6) 100 mol %.

mole fraction of 12−15 mol % in solution the surface is saturated with CPEO3. PM-IRRAS Measurements. While the contact angle analysis provides a physical parameter, i.e. the wettability of the surface, from which the SAM composition can be estimated, IR measurements allowed us to gather chemical information about the composition of the SAMs. Figure 4A shows polarization modulation-infrared reflection absorption spectra of SAMs assembled on reflective gold surfaces composed of 6MH and CPEO3. The SAM composed of pure CPEO3 shows characteristic absorption bands of the carbamate group at 1723 cm−1 (ν(CO)), 1537 cm−1 (δ(NH) + ν(C−N)), 1270 cm−1 (ν(CN) + δ(NH)) and 1124 cm−1 (ν(CO) + ν(CN)), which are in good agreement with the calculated ones.34 The ν(CH2) stretching modes of the cholesterol residue and ethoxy spacer can be observed in the range of 3000−2800 cm−1. The pure 6MH SAM PM-IRRAS spectrum shows asymmetric and symmetric ν(CH2) stretching modes below 3000 cm−1 from the alkyl chains. A ν(OH) stretching band, which would be expected at 3500−3100 cm−1, indicative of hydrogen bonding, cannot be observed.35 One explanation for this finding might be the non-well-ordered packing of short thiols resulting in a more random orientation of the OH groups. It is known that the structure of SAMs obtained from thiols with acyl chains shorter than C8−C10 is less ordered with a lower packing density compared to long chain thiols due to weaker interchain van der Waals interactions.36,37 PM-IRRAS spectra obtained from thiol mixtures ranging from 7 to 14 mol % CPEO3 in solution show absorption bands from both thiols. As a good indicator for the amount of CPEO3 8189

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angle and PM-IRRAS measurements, we conclude that the maximum occupancy of CPEO3 on the gold surface is reached with solutions containing 15−20 mol % of CPEO3, which is in good agreement with results reported by Jeuken et al.31 Considering the occupied surface area for a (√3 × √3)R30° based hexagonal packing per molecule on a gold [111] surface, the molar surface concentration of CPEO3 cannot be larger than 60%. This means that a SAM formed from a neat CPEO3 solution has the same CPEO3 surface concentration as a SAM formed from a 15−20 mol % CPEO3/6MH solution.21,38,39 The unoccupied binding sites between the CPEO3 molecules are filled with 6MH. Taking these considerations into account, the actual SAM composition can be approximated from the results of the contact angle and PM-IRRAS measurements. With this assumption, we can conclude that pore-spanning membranes on a SAM containing 20 mol % CPEO3 (4.8 mol % CPEO in solution) have a mean lateral tension of 2.3 mN m−1, while membranes on a SAM containing 30 mol % (7.1 mol % CPEO3 in solution) and 40 mol % (9.1 mol % CPEO3 in solution) CPEO3 show lateral tensions of 4.1 and 5.0 mN m−1, respectively. For SAMs with CPEO3 surface concentrations of more than 40 mol %, the stability of the pore-spanning membranes decreases considerably. These findings strongly support our hypothesis that the surface functionalization mainly influences the in-plane membrane tension. Besides this clear dependency of the lateral membrane tension on the CPEO3 surface concentration, the monitored membrane tensions show a rather broad distribution in particular for the binary SAM mixtures. One explanation for this observation might be the formation of partly phase-separated SAMs.40−42 An inhomogeneous distribution of CPEO3 on the surface, i.e. domain formation, would result in local variations of the resulting lateral membrane tension.

Figure 4. (A) PM-IRRAS spectra of SAMs composed of different ratios of 6MH and CPEO3. (B) Plot of the intensity of the ν(CO) band at 1723 cm−1 vs the mole fraction of CPEO3 in solution prior to the self-assembly process. A typical error bar is shown.

in the SAM, the maximum intensity of the band at 1723 cm−1 is plotted vs the mole fraction of CPEO3 in solution (Figure 4B). The intensity of the band increases with larger mole fraction of CPEO3 in solution, indicating that also the amount of CPEO3 in the SAM increases. This is in accordance with the increased hydrophobicity of the SAM as monitored by contact angle measurements. Saturation of the intensity of the IR band is reached at about 20 mol % CPEO3 in solution. Compared to the saturation value obtained from contact angle measurements and those reported by Jeuken et al.,31 this value is slightly larger. Moreover, the intensity curve obtained from the PM-IRRAS experiments is more strongly S-shaped in the range of 0−20 mol % CPEO3. These slight differences might be attributed to the different conditions under which the experiments have been performed. Contact angle measurements were performed in water; i.e., the hydrophobic CPEO3 molecules experience a high dielectric constant, while the PM-IRRAS measurements were done in air. These differences in the environment might result in slightly different orientations of the CPEO3 molecules on the surface. In particular, the PM-IRRAS signals are very sensitive to the orientation of the molecules on surface. If the dipole moment of the bond is parallel to the plane of incidence of the incoming light, a strong absorption is observed due to the surface-selection rules. The intensity decreases considerably if the bond tilts out of this plane. Consequently, the observed signal strongly depends on the orientation of the CPEO3 molecules on the surface. In particular, mixtures below 10 mol % CPEO3 show low CO stretching band intensities, suggesting a different molecular orientation. Correlation of Lateral Membrane Tension and Surface Functionalization. From our results obtained by contact

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CONCLUSIONS Pore-spanning lipid membranes suspending highly ordered pore arrays that are functionalized with mixed self-assembled monolayers (SAMs) composed of 6-mercapto-1-hexanol and the cholesterol derivative CPEO3 were probed by means of force spectroscopy to obtain their in-plane tension. AFM indentation experiments clearly demonstrate that the lateral membrane tension of the free-standing part of the membrane increases with increasing hydrophobicity of the SAM. Hence, the fine-tuning of the chemical composition of a SAM on the pore rims enables one to adjust the lateral tension of these artificial planar model membranes, which have been shown to allow addressing biologically relevant, tension-driven processes such as membrane fusion.43,44

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ASSOCIATED CONTENT

S Supporting Information *

Force volume images of pore-spanning membranes with corresponding force−distance curves. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.S.). Notes

The authors declare no competing financial interest. 8190

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the DFG (SFB 803).

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