pubs.acs.org/Langmuir © 2010 American Chemical Society
Mechanical Properties of Bare and Protein-Coated Giant Unilamellar Phospholipid Vesicles. A Comparative Study of Micropipet Aspiration and Atomic Force Microscopy Sabine Dieluweit, Agnes Csiszar, Wolfgang Rubner, Johannes Fleischhauer, Sebastian Houben, and Rudolf Merkel* Institute of Bio- and Nanosystems, Biomechanics (IBN-4), Research Centre Juelich, Germany Received February 4, 2010. Revised Manuscript Received March 17, 2010 In this study, protein-coated giant phospholipid vesicles were used to model cell plasma membranes coated by surface protein layers that increase membrane stiffness under mechanical or osmotic stress. These changed mechanical properties like bending stiffness, membrane area compressibility modulus, and effective Young’s modulus were determined by micropipet aspiration, while bending stiffness, effective Young’s modulus, and effective spring constant of vesicles were analyzed by AFM. The experimental setups, the applied models, and the results using both methods were compared here. As demonstrated before, we found that bare vesicles were best probed by micropipet aspiration due to its high sensitivity. The mechanical properties of vesicles with protein surface layers were, however, better determined by AFM because it enables very local deformations of the membrane with barely any structural damage to the protein layer. Mechanical properties of different species of coating proteins, here streptavidin and avidin, could be clearly distinguished using this technique.
Introduction Mechanical properties of biomembranes govern several biological processes, e.g., osmotic shrinkage and swelling of cells, cell membrane deformation during endo- and exocytosis, or cell migration.1 Interestingly, many cell membranes are able to adapt to their environment and change their composition or structure corresponding to necessity. Some archaea bacteria, for example, have an additional layer of proteins forming a two-dimensional crystal, called S-layer, coupled to the cytoplasmic membrane to protect the cell against osmotic pressure.2 This is achieved by the increased mechanical resistance, bending stiffness, and reduced elasticity of the crystalline protein layer on the membrane surface.3 In addition to S-layers, there are many other layer forming proteins like COPI,4 clathrin,5 or spam6 whose mechanical effects on membranes have been intensively investigated in recent years. Indeed, cellular studies on such proteins face the severe difficulty that several simultaneous and overlapping responses of the living organism have to be separated. To solve this, the application of model membrane systems has been established. Often, phospholipid-based giant unilamellar vesicles (GUVs) are adequate cell membrane models due to their composition, spherically closed structure, and size in the micrometer range. Moreover, they are filled with buffer instead of cellular components and biopolymers, and yield information exclusively about the mechanical properties of the membrane instead of the whole cell. An additional protein layer on their surface can mimic the influence *To whom correspondence should be addressed. E-mail: r.merkel@ fz-juelich.de. (1) McMahon, H. T.; Gallop, J. L. Nature 2005, 438, 590–594. (2) Engelhardt, H. J. Struct. Biol. 2007, 160, 190–199. (3) Xu, W.; Mulhern, P. J.; Blackford, B. L.; Jericho, M. H.; Firtel, M.; Beveridge, T. J. J. Bacteriol. 1996, 178(11), 3106–3112. (4) Manneville, J.-B.; Casella, J.-F.; Ambroggio, E.; Gounon, P.; Bertherat, J.; Bassereau, P.; Cartaud, J.; Antonny, B.; Goud, B. Prod. Natl. Acad. Sci. U.S.A. 2008, 105, 16946–16951. (5) Jin, A. J.; Prasad, K.; Smith, P. D.; Lafer, E. M.; Nossal, R. Biophys. J. 2006, 90(9), 3333–3344. (6) Cook, B.; Hardy, R. W.; McConnaughey, W. B.; Zuker, Ch. S. Nature 2008, 452, 361–364.
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of surface proteins, e.g., during osmotic stress,7 exo- and endocytosis, or cell adhesion. Several methods are available to determine the mechanical properties of biomembranes.8-11 Here, we compare two widely used techniques, micropipet aspiration and atomic force microscopy in force spectroscopy mode, with complementary application ranges for model biomembranes. Micropipet aspiration is an in situ, invasive method, where a small part of the membrane is aspirated by a glass pipet with a known suction pressure. With increasing suction pressure, the aspiration length increases. The change in total area of the membrane is proportional to the change in the projection length. In contrast, the tension acting in the entire membrane is proportional to the applied suction pressure. Mechanical parameters like bending stiffness12 or area dilatation modulus8 can be calculated using geometrical conditions, measured pressure, and aspiration length values. The technique has been also applied to cells13,14 and protein-coated giant unilamellar vesicles.15 Another frequently used and well-established method to probe mechanical properties of vesicles is atomic force microscopy in force spectroscopy mode.5,16,17 Briefly, a sharp tip on a soft cantilever is approached toward and retracted from the sample surface. The deflection of the cantilever is monitored as a function of its movement. From this, a force-distance curve can be (7) Csiszar, A.; Hoffmann, B.; Merkel, R. Langmuir 2009, 25, 5753–5761. (8) Kwok, R.; Evans, E. Biophys. J. 1981, 35, 637–651. (9) Lee, C.-H.; Lin, W.-C.; Wang, J. J. Opt. Eng. 2001, 40, 2077–2083. (10) Doebereiner, H.-G.; Gompper, G.; Haluska, Ch. K.; Kroll, D. M.; Petrov, P. G.; Riske, K. A. Phys. Rev. Lett. 2003, 91, 048301-1–048301-4. (11) Alonso, J.; Goldmann, W. H. Life Sci. 2003, 72(23), 2553–2560. (12) Evans, E.; Rawicz, W. Phys. Rev. Lett. 1990, 64(17), 2094. (13) Evans, E.; Waugh, R.; Melnik, L. Biophys. J. 1976, 16, 585–595. (14) Evans, E. Biophys. J. 1983, 43, 27–30. (15) Ratanabanangkoon, P.; Gropper, M.; Merkel, R.; Sackmann, E.; Gast, A. P. Langmuir 2003, 19(4), 1054–1062. (16) Liang, X.; Mao, G.; Ng, K. Y. S. J. Colloid Interface Sci. 2004, 278(1), 53–62. (17) Chen, Q.; Sch€onherr, H.; Vansco, G. J. Soft Matter 2010, 5, 4944–4950.
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detected. Whereas in micropipet aspiration global deformation of the vesicle is usually probed, AFM explores local membrane properties due to its superb spatial resolution. To explain the elastic response in AFM measurements, the extended Hertz model has been mostly applied.18-21 In that model, the deformation of two solid bodies pressed together, either two spheres or a cone and a planar material, is calculated. However, GUVs are fluid-filled shells rather than solid spheres; thus, a shell pressed between two plates is a more adequate description of a GUV deformed in between a tipless cantilever and the substrate. This model was developed and mainly applied for polyelectrolyte capsules22-25 and droplets of particle stabilized emulsions26 and, in some cases, for small protein-coated phospholipid vesicles.5 This paper demonstrates a parallel study of micropipet aspiration and AFM in force spectroscopy mode to determine mechanical material parameters like bending stiffness, effective Young’s modulus, and membrane area compressibility modulus of protein-coated phospholipid giant unilamellar vesicles. Such model membranes have frequently been used to mimic the role of surface proteins associated to cell plasma membranes in the case of mechanical or osmotic shocks. Even though, the proteins applied here, e.g., streptavidin and avidin, are not involved in mechanical osmoprotection of real cells, they drastically change the mechanical behavior of phospholipid membranes, as was shown earlier.7,15 These changed mechanical parameters were quantified by micropipet aspiration and AFM. The results were compared and their quality and suitability are discussed here.
Materials and Methods Chemicals. Model membranes were prepared using 1-stearoyl2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(cap biotinyl) (capBioDOPE) purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). SOPC and capBioDOPE were dissolved and mixed in chloroform at a molar ratio of 9:1 and a total lipid concentration of 1 mg/mL. The used proteins, streptavidin and avidin, with and without the fluorescent label Alexa Fluor488, were purchased from Invitrogen (Eugene, OR, USA). All other chemicals, like sucrose, glucose, 2-(N-morpholino)ethanesulfonic acid (MES), bovine serum albumin (BSA), and sodium bicarbonate (NaHCO3) buffer were obtained from VWR (Darmstadt, Germany). Vesicle Preparation. Giant unilamellar vesicles (GUVs) were prepared by the electroswelling technique.27 10 μL of lipid mixture (SOPC/capBioDOPE) was carefully deposited on indium tin oxide (ITO) coated glass slides. The lipid films were then dried under vacuum for at least 1 h. For electroswelling, the plates were placed in a chamber containing 2 mL of 300 mM sucrose solution (300 mosm/L) containing 10 μL of 0.5 M MES buffer (pH 5.5) or 10 μL of 0.1 M NaHCO3 buffer (pH 10.5). The ITO plates were separated with a 1 mm Teflon spacer. Vesicles were swollen applying an electric field at 1.5 V and 10 Hz for 2 h. Vesicle Coating with Streptavidin and Avidin. After preparation, vesicles were coated with proteins. For the preparation (18) Hertz, H. Journal f€ ur die reine und angewandte Mathematik 1882, 92, 156– 171. (19) Sneddon, I. N. Int. J. Eng. Sci. 1965, 3(1), 47–57. (20) Radmacher, M.; Fritz, M.; Hansma, P. K. Biophys. J. 1995, 69(1), 264–270. (21) Weisenhorn, A. L.; Khorsandi, M.; Kasas, S.; Gotzos, V.; Butt, H. J. Nanotechnology 1993, 2, 106. (22) Dubreuil, F.; Elsner, F.; Fery, A. Eur. Phys. J. E 2003, 12(2), 215–221. (23) Lulevich, V. V.; Vinogradova, O. I. Langmuir 2004, 20(7), 2874–2878. (24) Vinogradova, O. J. Phys.: Condens. Matter 2004, 16, R1105–R1134. (25) Elsner, N.; Dubreuil, F.; Weinkamer, R.; Wasicek, M.; Fischer, D.; Fery, A. Prog. Colloid Polym. Sci. 2006, 132, 117–123. (26) Ferri, J. K.; Carl, P.; Gorevski, N.; Russell, T. P.; Wang, Q.; Boker, A.; Fery, A. Soft Matter 2008, 4(11), 2259–2266. (27) Angelova, M. I.; Soleau, S.; Meleard, Ph.; Faucon, F.; Bothorel, P. Prog. Colloid Polym. Sci. 1992, 89, 127–131.
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Figure 1. Vesicle deformation by AFM. of crystalline streptavidin layers on GUVs, the pH value of the streptavidin solution was adjusted by 0.5 M MES buffer to pH 5.5 before adding this protein solution to the vesicle dispersion. The pH of the avidin solution was adjusted by 0.1 M NaHCO3 to pH 10.5 to avoid vesicle adhesion on the BSA-coated glass surface. In both cases, the osmotic pressure of protein solutions was adjusted to that of the vesicles. Vesicles were incubated in 70 μg/mL protein solution for at least 2 h. To prove complete surface coating with proteins, vesicles were prepared and coated with Alexa Fluor488 labeled streptavidin and avidin (Invitrogen, Eugene, OR, USA) (protein:protein-Alexa488 = 10:1). The fluorescence signal was analyzed by light microscopy (data not shown). Micropipet Aspiration. The experiments were carried out using a custom-designed hydrostatic pressure system (for details, see Supporting Information) combined with an inverted microscope (Axiovert 200, Carl Zeiss MicroImaging GmbH, Jena, Germany) with a water immersion objective. Experiments were carried out at 20 °C under isoosmotic conditions in an also custom-designed sample holder (see Supporting Information). Pipet and vesicle radii, the aspiration length, and the applied suction pressure values were recorded to calculate bending stiffness (κ) and membrane area compressibility modulus (KA). AFM Experiments. All experiments were carried out with a commercial AFM (NanoWizard from JPK, Berlin, Germany) combined with an inverted light microscope (Axiovert 200, Carl Zeiss MicroImaging, Jena, Germany). Standard silicon cantilevers with a nominal spring constant of 0.006 N/m (Biolever, Olympus Optical Co., Tokyo, Japan) were used to contact the vesicles seeded on BSA-coated glass surface (Figure 1). Round and smooth vesicles were selected in phase contrast microscopy. For coated vesicles, we also checked for the absence of undulations. Vesicle diameters were determined from light micrographs. Vesicles were positioned with respect to the cantilever and observed during the experiment under light microscopic control. The cantilever gently pressed on top of the vesicles with a speed of 1 μm/s. Vesicles were indented not with the tip of the cantilever but with the flat part behind the tip (Figure 1). Processing of AFM Measurements. The slope of the force vs tip-sample separation curve was linear fitted up to 1% indentation with respect to the diameter of the vesicle. According to Elsner et al.,25 the slope could be converted to elasticity, respectively, Eh2, E being the effective Young’s modulus of the membrane and h equivalent thickness (for details see Supporting Information).
Negative Stain Preparations and Transmission Electron Microscopy. A drop of sample was deposited onto a pioloformfilmed and carbon-coated EM grid. After several minutes, excess material was removed from the grid by a filter paper. The adsorbed vesicles were cross-linked with 2.5% glutaraldehyde solution in 0.1 M cacodylat buffer, pH 7.2 for 15 min, and negatively stained with 2% aqueous uranyl acetate solution for 10 min. Negative stain preparations were observed in a transmission electron microscope (TEM) (EM902 from Carl Zeiss, Oberkochen, Germany) with an acceleration voltage of 80 kV. All chemicals were purchased from Plano GmbH (Wetzlar, Germany).
Theoretical Considerations on Micropipet Aspiration. Bending stiffness and membrane area compressibility modulus of giant unilamellar vesicles (GUVs) were determined using the Langmuir 2010, 26(13), 11041–11049
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micropipet aspiration technique. A small part of the vesicle membrane was aspirated into a micropipet with a known suction pressure (ΔP). Under these conditions, the vesicle membrane is tensed and this surface tension (τ) was calculated as follows:12 ΔPRP τ ¼ RP 2 1RV
ð1Þ
where RP and RV denote the pipet and the vesicle radii, respectively. When the suction pressures (ΔP) were increased step by step, the aspiration length (ΔL) increased as a consequence of an elastic membrane response to the mechanical stress. The relative area expansion (R) has been given by Evans et al.:12 R ¼
i ΔA 1h = ðRP =RV Þ2 - ðRP =RV Þ3 ΔL=RP A0 2
ð2Þ
where ΔL represents the change of the aspiration length, A0 the membrane surface before stress application, and ΔA the area change. During the experiments, the applied suction pressures and the aspiration lengths were recorded. In the low tension range, the area expansion was dominated by smoothing of thermal undulations. Here, the bending stiffness (κ) resulted in a logarithmic dependence of relative area expansion on surface tension:28 τ 8πK R ð3Þ ln ¼ τ0 kB T kB is the Boltzmann constant and T the absolute temperature. In the high tension range, the changing area was mainly influenced by the membrane area compressibility modulus (KA) and increased linearly with tension:28 τ ¼ KA R
ð4Þ
Both limiting cases given above are connected by the relationship:12 kB T A τ ln 1 þ cτ þ R ¼ ð5Þ 8πK K KA where A is the entire projected area of the vesicle and c ∼ 0.1 a numerical constant of little impact. Subsequently, the effective Young’s modulus (E) of proteincoated vesicles was calculated using membrane bending stiffness also called membrane bending modulus (κ) and membrane thickness (h):29 E ¼
K12ð1 - ν2ves Þ h3
ð6Þ
Here, ν stands for the Poisson’s ratio of the vesicle membrane for which a value of 0.5 was assumed. Theoretical Considerations on AFM. When the AFMcantilever contacts the vesicle surface, the force, F(z), is given by FðzÞ ¼ kc de ¼ ks i
ð7Þ
where kc and ks represent the spring constants of cantilever and sample, respectively, de the cantilever deflection and i the indentation into the sample. Regardless of the sample geometry, the experiment can be considered as a combination of two linear springs according to Arnoldi et al.30 ks ¼
kc s 1-s
d ¼
RF
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 3ð1 - ν2 Þ 4Eh2
ð9Þ
Here, d denotes the deformation, R the radius of the spherical shell, h its wall thickness, ν the Poisson’s ratio, and E the Young’s modulus of the material. This model treats the special case of deformation of a thin elastic shell under point loads at the poles. In our experiments, we converted measured effective spring constants of the sample into Eh2. This way the derived values of the bending stiffnesses (cf. eq 6) depend only linearly on the estimate of the effective thickness h; see Discussion for more details. To compare these values with other data, the effective Young’s modulus was extracted by dividing the measured value Eh2 by h2. The Poisson’s ratio was assumed to be 0.5. The bending stiffness (κ) of protein-coated vesicles was derived from the effective Young’s modulus, or more exactly, Eh2, using eq 6.
Results Micropipet Aspiration. Micropipet aspiration yielded direct information about the bending stiffness (κ) and the area dilatation modulus (KA) of vesicles. From these, the effective Young’s modulus E could be calculated as given above. The membrane tension induced by suction was rising with the relative area expansion of the whole vesicle (Figure 2). In the low tension range, the area expansion was dominated by smoothing of thermal membrane undulations which was indicated by the linear dependence of relative area expansion on logarithm of surface tension (eq 3 and Figure 2a). In the high tension range, the area expansion was mainly influenced by the membrane area compressibility modulus (KA), which resulted in a linear dependency of tension and area expansion (eq 4 and Figure 2b). Results are summarized in Table 1. Bending stiffness of bare SOPC/capBioDOPE vesicles was found to be 0.4 10-19 J with a standard deviation of 0.1 10-19 J. The area dilatation modulus was found to be 176 mN/m with a standard deviation of 30 mN/m (Figure 3a). Compared to bare vesicles, streptavidin surface coating strongly influenced the mechanical parameters of the lipid bilayer. Their bending stiffness (1.1 10-19 J with a high standard deviation of 5.7 10-19 J) was found to be three times higher than that of SOPC/capBio-DOPE. Using this value and eq 6, their effective Young’s modulus were estimated at 9 MPa with a standard deviation of 46 MPa. Their area dilatation modulus was found to be 70 mN/m with a standard deviation of 40 mN/m, significantly lower than that of the bare vesicles with a similarly high standard deviation (Figure 3b). Surface coating with avidin resulted in an almost identical bending stiffness value (1.1 10-19 J with a significantly lower standard deviation of 1.4 10-19 J) compared to streptavidincoated vesicles. The measured area dilatation modulus of avidincoated vesicles (62 mN/m, standard deviation 20 mN/m) was in the same range as that of streptavidin-coated vesicles as well (Figure 3c).
ð8Þ
where s is the slope of the force-distance curve. (28) Rawicz, W.; Olbrich, K. C.; McIntosh, T.; Needham, D.; Evans, E. Biophys. J. 2000, 79, 328–339.
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Deformation of protein-coated vesicles could be described by the shell theory.29 To quantify the mechanical properties of the vesicles, the analytical solution of Reissner31,32 was applied
(29) Landau, L. D.; Lifschitz, E. M. Lehrbuch Der Theoretischen Physik, Elastizitaetstheorie; Akademie Verlag: 1991. (30) Arnoldi, M.; Fritz, M.; B€auerlein, E.; Radmacher, M.; Sackmann, E.; Boulbitch, A. Phys. Rev. E 2000, 62(1), 1034–1044. (31) Reissner, E. J. Math. Phys. 1946, 25, 80. (32) Reissner, E. J. Math. Phys. 1946, 25, 279.
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Dieluweit et al. Table 1. Mechanical Properties of Bare and Protein-Coated Vesicles as Determined by Micropipet Aspirationa sample
KA (mN/m)
s.d. KA (mN/m)
κ (10-19J)
s.d. κ (10-19 J)
E (MPa)
s.d. E (MPa)
n
SOPC/capBioDOPE vesicles 176 30 0.4 0.1 30 Streptavidin-coated SOPC/capBioDOPE vesicles 70 40 1.1 5.7 9 46 30 Avidin-coated SOPC/capBioDOPE vesicles 62 20 1.1 1.4 12 16 20 a Values are shown as mean. n represents the number of vesicles used during experiments. s.d. denotes the standard deviation. Distributions are shown in Figure 3.
Figure 2. (a) Logarithm of relative membrane tension (τ/τ0) vs relative area expansion (R = ΔA/A0) and (b) relative membrane tension (τ/τ0) vs relative area expansion (R = ΔA/A0) of a bare SOPC/capBioDOPE vesicle (squares), a streptavidin-coated (triangles), and an avidin-coated vesicle (circles) during micropipet aspiration. The following values of reference tension τ0 were used: 1.958 10-4 N/m for SOPC/capBioDOPE vesicles, 1.524 10-4 N/m for streptavidin-coated vesicles, and 1.147 10-4 N/m for avidin-coated vesicle.
AFM. Bare vesicles were also investigated by AFM in force spectroscopy mode. We observed force-distance curves with nonlinear parts right at the beginning of the contact and little reproducibility, indicating an unstable cantilever-sample interaction. Neither an exact identification of the contact point nor a linear fit could be performed. After retracting the cantilever from the vesicle, long filaments of lipids connecting cantilever and vesicle were produced. In the case of protein-covered vesicles, effective spring constants (ks) could be determined with AFM. From these, effective Young’s modulus (E) and bending stiffness (κ) were calculated. Figure 4a shows a representative force-distance curve consisting of trace and retrace as obtained from the measurement of a streptavidin-coated SOPC/capBioDOPE vesicle. Similar forcedistance curves were achieved from repeating indentations. Superposing trace and retrace revealed no significant differences between them indicating elastic membrane behavior, so plastic deformations and general hysteresis could be excluded (Figure 4a). Nevertheless, only advancing curves were examined and deformations up to 1% 11044 DOI: 10.1021/la1005242
of vesicle diameter were analyzed to make sure that the response of the vesicle was dominated by bending. For deformations up to 1% of vesicle diameter, a linear slope could be fitted (Figure 4b) to a force vs tip-sample separation curve. The resulting force-distance curves contained a combination of the spring constants of cantilever and sample. The effective spring constant of the sample is the initial slope of the force vs tip-sample separation curve without any transformation. The mean of all measured effective spring constants of streptavidincoated vesicles, ks, was 0.86 mN/m with a standard deviation of 0.74 mN/m. Here, 29 vesicles from 5 independent preparations and 234 force-distance curves were analyzed. For avidin-coated vesicles, the mean was 0.28 mN/m with a standard deviation of 0.15 mN/m (26 vesicles from 4 independent preparations and evaluating 176 force-distance curves). All later evaluations were based on these data. All results of AFM measurements are presented in Table 2. The value of Eh2 resulted directly from the force-measurement (eq 9). The mean of Eh2 for streptavidin-coated vesicles was 2.5 nN with a standard deviation of 2 nN; for avidin-coated vesicles, a mean of 1.0 nN with a standard deviation of 0.6 nN was measured. For increased comparability and clarity, the calculated effective Young’s modulus E was given as well. Eh2 was only converted by a factor, namely, divided by the squared layer thickness. For the layer thickness of streptavidin, a value of 4.8 nm was assumed,33-35 while for avidin, it is 4.3 nm.36-38 This yielded a mean of the effective Young’s modulus of 109 MPa with a standard deviation of 87 MPa for streptavidin-coated vesicles. For avidin-coated vesicles, the mean of the effective Young’s modulus was significantly lower and amounted to 53 MPa with a standard deviation of 30 MPa. The bending stiffness (κ) was calculated by using Eh2 in eq 6 which contains the Poisson’s ratio. This latter parameter was assumed as 0.5. The bending stiffness of streptavidin-coated SOPC/capBioDOPE vesicles was determined to be 1.3 10-18 J with a relatively high standard deviation of 1.1 10-18 J. The bending stiffness of avidin-coated SOPC/capBioDOPE vesicles was significantly lower and was found to be 4.7 10-19 J with a standard deviation of 2.6 10-19 J. The histograms of the bending stiffness of all streptavidin-coated and avidin-coated vesicles are shown in Figure 5a,b. To minimize the inaccuracy arising from the variation of spring constant and sensitivity of the cantilever, experiments with the same cantilever were consecutively executed on streptavidin- and avidin-coated vesicles within one day. The resulting bending stiffness values confirmed the significantly higher stiffness and (33) Hendrickson, W. A.; Paehler, A.; Smith, J. L.; Satow, Y.; Merritt, E. A.; Phizackerley, R. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86(7), 2190–2194. (34) Scheuring, S.; Mueller, D. J.; Ringler, P.; Heymann, J. B.; Engel, A. J. Microsc. 1999, 193(1), 28–35. (35) Weber, P.; Ohlendorf, D.; Wendoloski, J.; Salemme, F. Science 1989, 243 (4887), 85–88. (36) Green, N. M.; Joynson, M. A. Biochem. J. 1970, 118(1), 71–72. (37) Pinn, E.; P€aahler, A.; Sanger, W.; Petsko, G.; Green, N. M. Eur. J. Biochem. 1982, 123(3), 545–546. (38) Pugliese, L.; Coda, A.; Malcovati, M.; Bolognesi, M. J. Mol. Biol. 1993, 231(3), 698–710.
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Figure 3. Distribution of bending stiffness and area dilatation modulus of (a) bare SOPC/capBioDOPE vesicles (n = 30), (b) streptavidincoated (n = 30), and (c) avidin-coated vesicles (n = 20). Experiments were carried out using the micropipet aspiration technique. Table 2. Mechanical Properties of Bare and Protein-Coated Vesicles as Determined by AFMa κ
sample
-19
(10
J)
s.d. κ (10-19 J)
effective spring constant (mN/m)
s.d. effective spring constant (mN/m)
Eh2 (nN)
s.d. Eh2 (nN)
E (MPa)
s.d. E (MPa)
n
SOPC/capBioDOPE vesicles Streptavidin-coated SOPC/capBioDOPE vesicles 13 11 0.86 0.74 2.5 2.0 109 87 29 Avidin-coated SOPC/capBioDOPE vesicles 4.7 2.6 0.28 0.15 1.0 0.6 53 30 26 a Values are shown as mean. n represents the number of vesicles tested. s.d. denotes the standard deviation. Distributions are shown in Figure 5.
scatter of streptavidin-coated vesicles in comparison to avidincoated vesicles (see Figure 6). Negative Stain and Electron Microscopy of Protein Coated Vesicles. Due to drying and adsorption, vesicles collapsed on the electron microscopy grid surface. Concerning size variation of the vesicles, small vesicles also adsorbed on the grid. For clarity, Figure 7a shows a complete but smaller vesicle. The negatively stained streptavidin-coated vesicle presents deep membrane wrinkles due to collapse. At higher magnification, a regular lattice structure of crystallized streptavidin (Figure 7b,c) was observed. While the area of interconnected streptavidin patches was well visualized nearly on the whole vesicle, the borders of crystallized patches could not be localized due to the deep membrane wrinkles. Langmuir 2010, 26(13), 11041–11049
In contrast to the streptavidin crystal lattices on SOPC/ capBioDOPE vesicles, avidin layers displayed smooth surfaces without any regular pattern in between the membrane wrinkles (Figure 8a,b,c). Figure 8a shows a part of a collapsed GUV. An entire vesicle is not shown due to limited visual field. In the upper part of Figure 8a, the edge of the vesicle is observable. The darker components in the lower part are wrinkles caused by overlapping surplus membrane. Figures 7c and 8c illustrate the difference in the vesicle surfaces.
Discussion In this study, bare SOPC/capBioDOPE vesicles as well as streptavidin- and avidin-coated vesicles were used as model DOI: 10.1021/la1005242
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Figure 4. (a) Representative force-distance curve consisting of trace (black) and retrace (gray) as obtained from the measurement of a streptavidin-coated SOPC/capBioDOPE vesicle by AFM and (b) result of linear fit curve (white) to a tip-sample separation curve (black). The arrows show the limits of the fit range which extended from 0% to 1% vesicle diameter.
systems for living cell membranes. We focused on mechanical properties that were determined using micropipet aspiration and atomic force microscopy in force spectroscopy mode. Bare giant unilamellar vesicles are spherical, closed phospholipid bilayers, from SOPC and capBioDOPE lipids in our case. Their characteristic structure and size correspond to cell membrane structures and cell sizes as well. Similar to living cells, the average diameter of our giant unilamellar vesicles was within the range 10-20 μm. The thickness of a SOPC bilayer was reported to be 4 nm using X-ray diffraction.28 Addition of capBioDOPE does not influence this thickness, presumably, and was neglected here. In general, phospholipid bilayers successfully isolate the cell cytoplasm, or in our case the vesicle lumen, from its environment, but cannot effectively protect against strong mechanical stress. To increase the mechanical resistance of our vesicles, similar to some bacterial membranes, we attached a surface protein layer to the lipid bilayer. When bare SOPC/capBioDOPE vesicles were incubated in streptavidin or avidin solution, these proteins were bound to the vesicle surface by the strong and specific biotin streptavidin or biotin-avidin bonds. Streptavidin and avidin have four biotin binding pockets localized pairwise at two opposing sides of the protein. In our system, however, the protein can bind only two biotin molecules due to the sterical conditions as schematically shown in Figure 9. The biotin molecules were covalently bound to the DOPE component of the lipid bilayer through cap-tags. The cap-tag consists of 6 carbon atoms with an approximate length of 1 nm and allows a flexible coupling between lipid bilayer and protein shell. 11046 DOI: 10.1021/la1005242
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Figure 5. Distribution of bending stiffness of (a) streptavidincoated vesicles (n = 29) and (b) avidin-coated vesicles (n = 26) determined by AFM.
Figure 6. Results of measurements of bending stiffnesses of streptavidin-coated and avidin-coated GUVs performed on one day with the same cantilever.
We employed shell theory29,31,32 as mechanical model for protein-coated vesicles. In this theory, the behavior of hollow spherical objects formed from thin sheets of isotropic, elastic materials has been calculated. Protein-coated vesicles are micrometer-sized objects bound by layers of several nanometers thickness and thus well described as thin-walled shells. However, protein crystals (streptavidin) or highly viscous, perhaps even glass-like, protein layers (avidin)39-41 are certainly anisotropic materials responding differently to normal and tangential loads. Therefore, the calculated Young’s modules must be considered (39) Horton, M. R.; Reich, C.; Gast, A. P.; R€adler, J. O.; Nickel, B. Langmuir 2007, 23(11), 6263–6269. (40) Lou, C.; Wang, Z.; Wang, S. W. Langmuir 2007, 23(19), 9752–9759. (41) Fenz, S. F.; Merkel, R.; Sengupta, K. Langmuir 2008, 25(2), 1074–1085.
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Figure 7. TEM images of a negatively stained streptavidin-coated vesicle. (a) The vesicle collapsed on the electron microscope grid surface due to drying and adhesion. Its surface underwent membrane folding processes. Scale bar = 500 nm. (b) Higher magnification reveals an overall crystalline lattice pattern on the vesicle surface. Scale bar = 100 nm. (c) Zoom out of Figure 7b smoothed with a Gaussian filter of 0.25 nm radius and gray scale aligned. Scale bar = 50 nm.
Figure 8. TEM images of a negatively stained avidin-coated vesicle. (a) Similar to streptavidin-coated vesicles, avidin-coated lipid shells collapsed on the supporting EM grid forming surface wrinkles. On this micrograph, only a part of a vesicle is shown. Scale bar = 5 μm. (b) Its surface was smooth; no regular pattern was observed. Scale bar = 200 nm. (c) Zoom out of (b) smoothed with a Gaussian filter of 0.25 nm radius and gray scale aligned. Scale bar = 50 nm.
Figure 9. Schematic structure of a streptavidin-coated phospholipid bilayer, where the streptavidin molecules are attached to the SOPC lipid bilayer by specific binding of two biotin functionalized lipid molecules (capBioDOPE).
with care. They are equivalent values, especially because we used the structural size of the proteins as mechanical layer thickness. Since the stiffness of the protein layer is most likely much higher than that of the lipid bilayer and a fluid lipid bilayer cannot support static shear stress, the thickness of the lipid bilayer was neglected for the calculation of the overall mechanical response. Langmuir 2010, 26(13), 11041–11049
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Only the thickness of the protein layer was considered. For the layer thickness of streptavidin, a value of 4.8 nm was assumed,33-35 and for avidin, 4.3 nm.36-38 The successful surface coating of vesicles with proteins was controlled using fluorescent-labeled proteins. The overall homogeneous fluorescent signal on the vesicle surface indicated continuous protein coating in both cases. Furthermore, deformation according to shell theory occurs with varying enclosed volume of the shell. Thus, water flows through the membrane as discussed for this system in reference.7 Thus, in principle, resistance against water flow could play a role for the effective stiffness of the vesicles in AFM indentation. However, this can only occur if the osmolarity of the inner medium exceeds that of the outer one. From basic geometrical considerations, we conclude that the relative volume change of a shell indented by 1% of its diameter amounts to, at most, 3 10-4. Accordingly, osmotic pressure can be increased at most by this fraction, in our case, 0.09 mosm/L. Due to evaporation from the AFM chamber, the osmolarity of the outer solution was increasing with a rate of approximately 10 mosm/(Lh). As the osmolarities of inner and outer solution were initially identical, osmotic resistance could only have played a role during the first minute of the experiment. In line with these arguments, we did not observe any correlation of effective stiffness in AFM indentation with age of the sample (up to one hour). On the basis of these considerations, protein-coated vesicles have been approximated by isotropic elastic shells to analyze our measured values. As also demonstrated before,4,8,13,14,28 we found that micropipet aspiration is an appropriate method to investigate bare phospholipids GUVs. Their bending stiffness as well as area dilatation (Table 1) were in close agreement with literature values determined by other techniques.16,42 Although the aspiration of a small part of the membrane into a glass pipet is an invasive step, the homogeneous structure of a phospholipid bilayer was not destroyed by the pipet due to its two-dimensional fluid character. A comparison of results of micropipet aspiration with those of AFM in force spectroscopy mode shows marked differences. Micropipet aspiration of streptavidin- and avidin-coated vesicles yielded significantly lower bending stiffness and effective Young’s modulus values than the AFM technique (see Tables 1 and 2). Additionally, the standard deviations of our micropipet aspiration measurements on protein-coated vesicles were remarkably high. Moreover, Domke et al.43 specified the elasticity of surface proteins in the range of 100 MPa to GPa, while our values were at least an order of magnitude lower. Furthermore, in micropipet aspiration the two proteins endowed the membrane with similar resistance against bending and stretching. This seemingly contradicts the findings of our earlier work where different mechanical properties of streptavidin and avidin layers were demonstrated.7 To explain this gap between our measured values and literature data, the experimental conditions have to be thoroughly discussed here. First of all, the contact areas (the sucked membrane area by micropipet aspiration and the squeezed membrane area by AFM) were approximately in the same range. In Figure 10, schematic diagrams of the two methods used to examine the vesicles are shown. In AFM, in force spectroscopy mode the crystalline shell of the vesicle is bent locally close to the contact points of the poles (Figure 10a). The protein layer remained intact. Repeated indentation of an individual vesicle demonstrated the reversibility of this measurement. In contrast, micropipet aspiration caused (42) Niggemann, G.; Kummrow, M.; Helfrich, W. J. Phys. II 1995, 5, 413–425. (43) Domke, J.; Rotsch, C.; Hansma, P. K.; Jacobson, K.; Radmacher, M. Scanning Probe Microsc. Polym. 1998, 178–193.
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Figure 10. Schematic diagrams of vesicle deformation by both methods used: (a) shows the mechanical model to describe the AFM experiment, i.e., a protein-coated vesicle between two plates; (b) shows a protein-coated vesicle locally damaged by micropipet aspiration; ΔP is the suction pressure.
plastic deformation of the protein shell (Figure 10b).15 The mechanical deformation was transferred to the whole vesicle, so tension is predominant.15 All established relations for micropipet aspiration experiments depend on membrane fluidity. A crystalline streptavidin layer on top of a fluid membrane results in shear stiffness, plastic deformation, and nonspherical shape of the free membrane part.15 Thus, eqs 1-4 are of doubtful value for streptavidin-coated vesicles. Moreover, micropipet aspiration measurements of membrane bending stiffness rely on smoothing the thermal undulation of the membrane. However, membrane rigidification by an additional protein layer resulted in a strong reduction of membrane undulations and, correspondingly, in a much lower reservoir of “hidden” area, that could be accessed by micropipet aspiration. This can be seen mathematically, e.g., in eq 3 which indicates an inverse relation between relative area expansion R at constant tension τ and bending stiffness κ. Second, it is highly probable that the micropipet drastically damages the lateral homogeneity and structure of the protein shell on the whole suction surface, ca. 10-100 μm2, during the aspiration process15 as can be seen in Figure 10b. Here, the measured values describe more the locally damaged material than the overall material properties. Therefore, the calculation of the effective Young’s modulus yielded unrealistic values and extreme scatter. Besides micropipet aspiration, AFM in force spectroscopy mode was chosen to investigate the same membrane systems. AFM analysis of bare SOPC/capBioDOPE giant unilamellar vesicles was complicated by several experimental challenges. For example, the analysis of the force-distance curve has been very sensitive to the definition of the contact point between cantilever and vesicle. Vesicle membrane fluctuations in the micrometer range led to an imprecise identification of the contact point. Moreover, for bare vesicles it is not valid to assume a spherical vesicle shape, which is, however, indispensable to apply the above-mentioned model. Another main challenge arises from nanotube formation between cantilever and membrane. After cantilever contact with the vesicle top, lipid stuck to the cantilever surface and the retracting cantilever exerted stretching forces on the membrane. On the basis of the low bending stiffness of SOPC of about 11048 DOI: 10.1021/la1005242
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0.4 10-19 J, the formation of lipid nanotubes could not be avoided. Therefore, only one vesicle could be deformed per cantilever, which made these investigations highly arduous. The phenomenon has been described before.44,45 Due to the experimental obstacles described above, AFM studies on bare GUVs remained without satisfying results. In contrast to noncoated lipid shells, protein-coated vesicles were successfully investigated with AFM technique. The main difference of mechanical deformation performed by AFM in contrast to micropipet aspiration is that the AFM cantilever locally bends the protein crystal shell near the cantilevermembrane contact point. If this local deformation is in the range of 1% of vesicle diameter, the deformation remains reversible. The reversible character has been controlled by analysis of the retrace curves. The crucial result obtained by AFM was the difference in mechanical parameters between avidin- and streptavidin-coated vesicles. The effective spring constant, Eh2, effective Young’s modulus, and bending stiffness of streptavidin-coated vesicles were 2-3 times higher than for avidin-coated vesicles. Data taken at one and the same day and therefore with identical calibrations (Figure 6) showed a difference in mechanical parameters between both proteins even more pronounced than the pooled data. Therefore, this difference is highly significant. It can be explained by the different structures of both protein layers. Streptavidin layers crystallized as the outermost layer of the vesicle, as presented in the TEM image in Figure 7, while avidin did not exhibit a regular structure (Figure 8). Additionally, investigated by AFM streptavidin-coated vesicles showed a higher scatter than avidin-coated vesicles. Even this was related to the protein layer structure. Presumably, it was significant where the cantilever hit the vesicles, whether on a continuous crystal patch or on the boundary of patches. Ratanabanangkoon et al.46 imaged different lattice directions of streptavidin crystals on vesicles, depicting such boundaries. In contrast, avidin-coated vesicles were covered more homogeneously. A comparison with literature values on Young’s modulus of protein crystals reveals that our data (110 MPa for streptavidin coating and 53 MPa for avidin coating) are in the lower range of reported values (100 MPa to 1 GPa).43 This is most likely due to the fact that we studied monolayers of proteins where some of the protein-protein contacts that stabilize three-dimensional bulk systems are not present. Moreover, packing defects are very important especially in the case of the crystalline streptavidin layer. In mammalian cells, clathrin-coated vesicles display a structure related to our system. They consist of a phospholipid membrane coated with a layer of the protein clathrin that is also forming a crystal-like structure. The bending stiffness of these vesicles was studied by Jin et al.5 using AFM. These authors used a mechanical model that takes into account bending stiffness of the shell and neglects other deformation modes like in-plane shear. This model was originally developed for vesicles from fluid lipid bilayers47 and yielded a bending stiffness of 11 10-19 J, very close to the value of 13 10-19 J for streptavidin-coated vesicles found here and somewhat above the value of 4.7 10-19 J found for avidincoated vesicles. The similar stiffnesses of streptavidin-coated (44) Maeda, N.; Senden, T. J.; di Meglio, J.-M. Biochim. Biophys. Acta, Biomembr. 2002, 1564(1), 165–172. (45) Pera, I.; Stark, R.; Kappl, M.; Butt, H.-J.; Benfenati, F. Biophys. J. 2004, 87(4), 2446–2455. (46) Ratanabanangkoon, P.; Gast, A. P. Langmuir 2002, 19(5), 1794–1801. (47) Helfrich, W. Z. Naturforsch. 1973, 28, 693–703.
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Figure 11. Overview of the comparative study of micropipet aspiration and AFM techniques to determine some mechanical properties of protein-coated and bare giant unilamellar phospholipid vesicles presented in this paper.
vesicles and clathrin-coated vesicles most likely originate from the very similar architecture of both systems.
model membrane system of such character, like lipid membranes with different lipid composition or even lipid shells with channel protein patches, can be studied by micropipet aspiration. In contrast to these two-dimensional fluid membranes, lipid bilayers combined with a crystalline shell, e.g., a surface protein shell, are not appropriate for micropipet investigation due to its irreversible damaging effect on the crystalline shell. AFM in force spectroscopy mode is also suitable for the study of bare vesicles (Figure 11). In practice, strong vesicle fluctuations and nanotube formations between vesicles and AFM-cantilever made these measurements cumbersome and therefore not recommendable. In contrast, the mechanical properties of vesicles with a coupled protein surface layer could be better determined by AFM. Even different proteins like streptavidin and avidin could be distinguished accurately with this technique.
Conclusion
Acknowledgment. The authors thank U. Seifert for enlightening discussions on the shell theory. Export technical support by the mechanics work shop of IBN was crucial for this project.
The mechanical properties of giant unilamellar vesicles can be determined using micropipet aspiration and atomic force microscopy in force spectroscopy mode. However, both methods are suited for vesicles with different mechanical behavior, bare vesicles can be successfully probed by micropipet aspiration due to their two-dimensional fluid character. Theoretically, every
Supporting Information Available: Details of the experiment of micropipet aspiration technique and AFM in force spectroscopy mode and processing of AFM measurements. This material is available free of charge via the Internet at http://pubs.acs.org.
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