Solid Supported Membranes Doped with PIP2: Influence of Ionic

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Solid Supported Membranes Doped with PIP2: Influence of Ionic Strength and pH on Bilayer Formation and Membrane Organization Julia A. Braunger,† Corinna Kramer,† Daniela Morick, and Claudia Steinem* Institut für Organische und Biomolekulare Chemie, Georg-August Universität, Tammannstr. 2, 37077 Göttingen, Germany S Supporting Information *

ABSTRACT: Phosphoinositides and in particular L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) are key lipids controlling many cellular events and serve as receptors for a large number of intracellular proteins. To quantitatively analyze protein−PIP2 interactions in vitro in a time-resolved manner, planar membranes on solid substrates are highly desirable. Here, we describe an optimized protocol to form PIP2 containing planar solid supported membranes on silicon surfaces by vesicle spreading. Supported lipid bilayers (SLBs) were obtained by spreading POPC/PIP2 (92:8) small unilamellar vesicles onto hydrophilic silicon substrates at a low pH of 4.8. These membranes were capable of binding ezrin, resulting in large protein coverage as concluded from reflectometric interference spectroscopy and fluorescence microscopy. As deduced from fluorescence microscopy, only under low pH conditions, a homogeneously appearing distribution of fluorescently labeled PIP2 molecules in the membrane was achieved. Fluorescence recovery after photobleaching experiments revealed that PIP2 is not mobile in the bottom layer of the SLBs, while PIP2 is fully mobile in the top layer with diffusion coefficients of about 3 μm2/s. This diffusion coefficient was considerably reduced by a factor of about 3 if ezrin has been bound to PIP2 in the membrane.

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means of fluorescence microscopy.16 Although SSMs are characterized by the lack of a large internal compartment as compared to unilamellar vesicles, they have the advantage of being planar and long-term stable.17,18 Thus, they are accessible by a number of surface sensitive methods such as ellipsometry,19,20 electrical impedance spectroscopy,21 fluorescence microscopy, and atomic force microscopy,22 which allow monitoring their integrity and biophysical properties as well as their lateral organization. They are also versatile tools to monitor protein−lipid interactions in a quantitative manner.23,24 Methods based on unilamellar vesicle spreading have been developed to generate SSMs either on hydrophobic monolayers resulting in hybrid lipid membranes or on hydrophilic substrates such as glass, mica, or silicon.25 Many different factors influence the unilamellar vesicle spreading process leading to continuous membranes that cover large surface areas including ionic strength, vesicle size and composition, pH value, and surface charge.20,26−28 While the vesicle spreading process appears to be rather robust on hydrophobically functionalized substrates,29 the formation of continuous bilayers on hydrophilic substrates is strongly influenced by the above-mentioned parameters.

INTRODUCTION Although L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) constitutes only a minor fraction of about 1% of the lipids in the plasma membrane, it fulfills a multiplicity of functions within the cell including cytoskeletal attachment,1 regulation of actin polymerization,2 enzyme and ion channel activation,3,4 and endo- and exocytosis.5−7 Moreover, PIP2 is the precursor compound of three second messengers, namely diacylglycerol, inositol-1,4,5-trisphosphate (IP3), and phosphatidylinositol3,4,5-trisphosphate (PIP3), thus participating in signal transduction.8 In general, in vivo experiments investigating the impact of PIP2 at the plasma membrane are rather challenging as the PIP2 metabolism is controlled by a complex network of enzymatic processes.9 Unraveling parts of such a complex interplay can be achieved by extracting single components and rebuilding a membrane system of known composition. Such artificial membranes have emerged as versatile tools to study membrane-confined biological processes in a well-defined environment, while retaining some of their important properties such as two-dimensional fluidity. Several biophysical studies employing planar solid supported membranes (SSMs), 1 0−1 2 large unilamellar vesicles (LUVs),13,14 and giant unilamellar vesicles (GUVs)15,16 aimed at elucidating interactions between proteins and PIP2 under defined conditions. Considering dimensions and membrane curvature, micrometer-sized GUVs rather mimic cells than LUVs (∼100 nm in diameter) and are easily investigated by © 2013 American Chemical Society

Received: July 12, 2013 Revised: October 21, 2013 Published: October 21, 2013 14204

dx.doi.org/10.1021/la402646k | Langmuir 2013, 29, 14204−14213

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vesicles and the substrate by either screening or reducing the negative charges. Screening of charges can be achieved by increasing the ionic strength. Monovalent cations such as Na+ and K+ reduce the Debye length and thus may favor the formation of a uniform bilayer.33 As it has been shown that Na+ binds more strongly to the phosphate moieties of phospholipids than K+ does,34 we replaced KCl in buffer A by NaCl and at the same time increased the concentration to 200 mM. Another additive often used to increase the success rate in vesicle spreading is calcium ions.25,35,36 Even though the spreading process in the presence of Ca2+ is still a matter of discussion, it is assumed that during the initial phase of vesicle adsorption, Ca2+ bridges the phosphate and/or carbonyl groups of the lipids with the silicon surface.37,38 Moreover, local dehydration and defects in lipid packing evoked by Ca2+ are suggested.39 Local stress induced by Ca2+ may lead to an increased exposure of the hydrophobic core, giving rise to enhanced hydrophobic attraction and hence vesicle spreading.40 In our experiments, 2 mM CaCl2 was added to buffer A. Another strategy we pursued was to reduce the negative charges of the PIP2 molecules by lowering the pH value. Within a pH range of 6.8−7.7, the net charge of PIP2 is calculated to be −4, whereas it is reduced to about −3 at pH 4.8.41,42 In addition, the negative surface charge of the silicon substrate is reduced by a factor of 3.43 We used a buffer composition of 20 mM citrate, 50 mM KCl, 0.1 mM EDTA, and 0.1 mM NaN3, pH 4.8. By means of reflectometric interference spectroscopy (RIfS), we monitored the change in optical thickness (ΔOT) during the spreading process of SUVs composed of POPC/PIP2 (92:8) on hydrophilized silicon substrates under the given different buffer conditions. The subsequent binding of ezrin to PIP2 was then performed after exchange to buffer A for all membrane preparations. As depicted in Figure 1A1, the addition of POPC/PIP2 (92:8) SUVs in Ca2+-containing buffer A led to an increase in optical thickness up to a maximum value of ΔOT = 10.2 nm. Taking the refractive index of a bilayer with nlipid = 1.5 into account,44 ΔOT translates into a membrane thickness tm = 6.8 nm. This value is significantly larger than that determined for a POPC bilayer (tm = 3.98 ± 0.08 nm) by means of small-angle neutron and X-ray scattering.45 To remove Ca2+ after the spreading process, the system was flushed with buffer A containing 10 mM EGTA. This resulted in a decrease of ΔOT = −1.9 nm, which translates in a final ΔOT = 8.3 nm for the bilayer (tm = 5.5 nm), which is still larger than expected. To gather information on whether the PIP2 receptor lipid is capable of binding a PIP2 binding protein under these conditions, 0.7 μM of ezrin was added, a concentration that is well in the saturation regime of the adsorption isotherm.12 Unexpectedly, almost no ezrin binding was monitored by RIfS (Figure 1A2). From four independent experiments, an average ΔOT = 0.6 ± 0.9 nm was obtained after ezrin addition (Figure 2). Spreading POPC/PIP2 (92:8) SUVs in buffer A, in which 50 mM KCl was exchanged against 200 mM NaCl, resulted in an increase in optical thickness of ΔOT = 6.4 nm (tm = 4.3 nm, Figure 1B1). This value is closer to that of a POPC lipid bilayer being about 10% larger than the exact value of a pure POPC bilayer, which might in part be a result of the PIP2 molecules protruding about 0.6 ± 0.1 nm from the membrane surface.46 After the spreading process was finished, the buffer was exchanged against buffer A, affecting ΔOT only to a very minor extent. The addition of 0.7 μM ezrin resulted in protein binding

Here, we describe that the buffer composition greatly influences the vesicle spreading process and the resulting SSMs containing the lipid PIP2. Owing to its phosphate groups, PIP2 possesses a net negative charge ranging from −3 to −5 depending on the buffer conditions. The pKa values of the phosphate groups at the 4′ and 5′ position of the inositol ring are approximately 6.7 and 7.7.30−32 The net negative charge can result in electrostatic repulsion between PIP2 containing vesicles and the negatively charged silicon substrate,27,28 thus preventing continuous lipid bilayer formation at pH 7.4. The point of zero charge of silicon surfaces ranges between pH 1.0− 3.7 depending on the ionic strength.30,31 We elucidated different buffer conditions to spread PIP2 containing lipid vesicles on hydrophilic silicon substrates and analyzed the accessibility of the PIP2 head groups for protein binding, making use of the PIP2 binding protein ezrin. By comparing the diffusive behavior of PIP2 in supported lipid bilayers (SLBs) with that of PIP2 only being in the top monolayer (supported hybrid membranes, SHMs), we were further able to show that PIP2 in the surface-facing bottom monolayer is almost fully immobile. The corresponding membrane architectures are depicted in Scheme 1. Scheme 1. Schematic Drawing of the Two Membrane Systems on Silicon Support Used in This Studya

a

SUV: small unilamellar vesicle; SLB: supported lipid bilayer; SHM: supported hybrid membrane.

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RESULTS AND DISCUSSION RIfS Analysis of Vesicle Spreading and Ezrin Binding. While spreading of small unilamellar vesicles (SUVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ PIP2 in 20 mM Tris/HCl, 50 mM KCl, 0.1 mM EDTA, 0.1 mM NaN3, pH 7.4 (buffer A) on hydrophilic silicon substrates allows the reproducible formation of continuous bilayers with high surface coverage up to a maximum PIP2 concentration of 4 mol % (Supporting Information, Figure S1), it turned out that larger PIP2 concentrations prevented bilayer formation under the given conditions. Even though PIP2 concentrations of more than 4 mol % exceed that found in most natural membranes, some PIP2-protein binding studies require larger PIP2 surface concentrations to ensure a good signal-to-noise ratio. Thus, we elucidated different conditions to be able to produce continuous lipid bilayers composed of POPC doped with 8 mol % PIP2 on silicon substrates and investigated whether the PIP2 headgroup is accessible for protein binding. Conditions were chosen to minimize electrostatic repulsion between lipid 14205


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Figure 1. Kinetics of SUV adsorption and spreading on hydrophilic silicon wafers followed by the adsorption of ezrin monitored by RIfS. SUVs were composed of POPC/PIP2 (92:8). Three different spreading conditions were chosen: (A) buffer A supplemented with 2 mM CaCl2; (B) buffer A, in which 50 mM KCl were replaced with 200 mM NaCl; (C) 20 mM citrate, 50 mM KCl, 0.1 mM EDTA, and 0.1 mM NaN3, pH 4.8. (1) The addition of 0.2 mg/mL SUVs resulted in a time-dependent change in OT. The time points of vesicle addition and rinsing with buffer A as well as EGTAcontaining buffer A (buffer A + 10 mM EGTA) when necessary are marked with dashed lines. (2) After rinsing with buffer A, 0.7 μM ezrin was added and the time course in ΔOT was monitored. The OT value was set to zero to allow for a quick determination of ΔOT upon protein binding.

Fluorescence Analysis of SLBs and Ezrin Binding. To inspect the resulting SLBs, fluorescence microscopy images were taken before and after the addition of ezrin. While ezrin was labeled with AlexaFluor488, the membranes were doped with the fluorophore perylene to avoid major crosstalk of the two fluorophores. For membranes prepared in Ca2+-containing buffer A a blue perylene fluorescence was visible throughout the substrate (Figure 3A1), which we interpret as a fluorescently labeled bilayer. Bright fluorescent circular structures were discernible, which we attribute to attached lipid material, and which was also found throughout the sample. The heterogeneous fluorescence of a lipid bilayer with attached lipid material became better visible by using the fluorophore β-Bodipy-C12HPC (Supporting Information, Figure S2). This material could only partly be detached from the bilayer, even after Ca2+ removal (Supporting Information, Figure S2), thus explaining the larger membrane thickness of tm = 6.8 nm (before Ca2+ removal) and 5.5 nm (after Ca2+ removal) as determined by RIfS. The addition of 0.5 μM AlexaFluor488 labeled ezrin resulted only in small submicrometer-sized protein clusters on the surface (Figure 3A2), which explains the small change in ΔOT monitored by RIfS. A surface coverage of 7 ± 2% was roughly estimated from the fluorescence micrographs by pixel analysis. SLBs prepared in the presence of 200 mM NaCl also showed a continuous blue perylene fluorescence indicating the formation of a continuous lipid bilayer with large surface coverage on the silicon substrate (Figure 3B1), in agreement with the observed membrane thickness of tm = 4.3 nm. The addition of 0.5 μM AlexaFluor488 labeled ezrin led to few larger protein clusters with diameters of several micrometers among many submicrometer-sized clusters. The slightly brighter blue fluorescence (Figure 3B1) matching the structures of the Alexa488 labeled ezrin clusters (Figure 3B2) is a result of a small crosstalk between the two fluorophores. On average, a

Figure 2. Change in optical thickness (ΔOT) after addition of 0.7 μM ezrin in buffer A (hatched bars) and after rinsing with buffer A (solid bars) depending on the vesicle spreading conditions (2 mM CaCl2: buffer A supplemented with 2 mM CaCl2, 200 mM NaCl: buffer A, in which 50 mM KCl were replaced with 200 mM NaCl, and pH 4.8: 20 mM citrate, 50 mM KCl, 0.1 mM EDTA, 0.1 mM NaN3, pH 4.8). The error bars are the standard deviation of the mean.

with an increase in ΔOT = 1.0 nm (Figure 1B2). Part of the proteins was reversibly bound to the PIP2 containing membrane indicated by the decrease in ΔOT = −0.6 nm upon rinsing with buffer A. From three different experiments, an average ΔOT = 1.4 ± 0.7 nm was determined after protein addition, which slightly decreased to 1.0 ± 0.4 nm after buffer A rinsing as shown in Figure 2. In the presence of citrate buffer, pH 4.8, POPC/PIP2 (92:8) SUVs also spread on the silicon substrate to form a bilayer as deduced from the increase in optical thickness with a maximum ΔOT = 6.3 nm (tm = 4.2 nm). Buffer exchange to buffer A did not affect the optical thickness (Figure 1C1). However, in contrast to the results obtained for protein binding under the two other chosen conditions, the addition of 0.7 μM ezrin led to a significant increase in ΔOT = 6.5 nm. Rinsing with buffer A resulted in a partial desorption of the protein resulting in ΔOT = −1.8 nm (Figure 1C2). The average change in optical thickness was determined to be ΔOT = 6.4 ± 0.6 nm, which reduced to 5.1 ± 0.7 nm after buffer A rinsing (n = 6) (Figure 2). 14206


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From the presented results it becomes obvious that the binding capability of PIP2 is altered by the buffer conditions used for vesicle spreading, even though the buffer conditions for protein binding (buffer A) were identical in all cases. We hypothesized that the different binding capacities of the SLBs are a result of an inhomogeneous distribution of the PIP2 molecules in the membrane, which would make part of the PIP2 molecules inaccessible if some ezrin has already been bound. In addition, in the case of the Ca2+ conditions, the attached lipid material can block PIP2 binding sites. That attached lipid material partly blocks PIP2 bindings sites was corroborated by an experiment, where almost no vesicles were bound to the membrane, but the membrane was treated with CaCl2. In this case, more ezrin bound to the membrane than in case of membranes prepared in the presence of Ca2+ at pH 7.4, but still less than on membranes prepared at pH 4.8 without Ca2+ treatment (Supporting Information, Figure S3). To analyze whether the observed differences in protein binding capacities of the SLBs are a result of an altered lateral organization of PIP2 in the membrane, we investigated the PIP2 distribution in the SLBs making use of fatty acid labeled PIP2 (Bodipy TMR-PIP2 C16). PIP2 Distribution in SLBs. Fluorescence images of SLBs with fluorescently labeled PIP2 are shown in Figure 4. The fluorescence images clearly show that the PIP 2 distribution within the membrane is greatly influenced by the chosen buffer conditions. SLBs prepared in the presence of Ca2+ exhibited a large number of attached lipid material, which could not be removed by depleting the solution of Ca2+ as already discussed. Despite these bright spots, which make it difficult to obtain information about the PIP2 distribution within the underlying lipid bilayer, the PIP2 fluorescence distribution appears to be inhomogeneous (Figure 4A2). SLBs obtained from spreading PIP2 containing POPC vesicles in the presence of 200 mM NaCl also showed a very inhomogeneous PIP2 fluorescence (Figure 4B2), whereas spreading PIP2 containing POPC vesicles at pH 4.8 resulted in a more homogeneous PIP2 fluorescence (Figure 4C2). This result suggests that under low pH conditions the PIP2 molecules are more homogeneously distributed resulting in large ezrin coverage, while the ezrin coverage is significantly reduced if PIP2 is more inhomogeneously distributed. Attached lipid material in conjunction with inhomogeneously distributed PIP2 almost fully prevents ezrin binding. The inhomogeneous distribution of the PIP2 fluorescence intensity in the membrane might be attributed to PIP2 clusters. The presence of PIP2 clusters in lipid monolayers in presence of Ca2+ has been reported previously.47,48 By means of atomic force microscopy images and transmission electron microscopy images of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers containing different amounts of PIP2 (1−50 mol %) in the presence of 1 mM Ca2+, Wang et al.48 were able to observe PIP2-rich clusters. Levental et al.47 determined a dissociation constant of about 3 μM for Ca2+ binding to PIP2 in pure PIP2 monolayers at the air−water interface. Ca2+-induced PIP2 clusters were also reported in GUVs.13,16 Ellenbroek et al.49 were able to show experimentally and in coarse-grained simulations that PIP2 forms clusters already at low molar fractions of 2% mediated by Ca2+ bridging of the phosphate groups. While in GUVs and monolayers at the air−water interface cluster formation was reversible, i.e., Ca2+ removal by EDTA or EGTA (Kd = 0.13 μM at pH 7.1)50

Figure 3. Fluorescence images of POPC/PIP2/perylene (91:8:1) SLBs on hydrophilic silicon wafers. Three different spreading conditions were chosen: (A) buffer A supplemented with 2 mM CaCl2; (B) buffer A, in which 50 mM KCl were replaced with 200 mM NaCl; (C) 20 mM citrate, 50 mM KCl, 0.1 mM EDTA, 0.1 mM NaN3, pH 4.8. Before protein incubation, all SLBs were rinsed with buffer A. In the case of Ca2+ promoted spreading, SLBs were additionally rinsed with buffer A containing 10 mM EGTA after bilayer formation. (1) Perylene fluorescence images of the SLBs. (2) Fluorescence images of AlexaFluor488 labeled ezrin. 0.5 μM of fluorescently labeled ezrin was added, and fluorescence images were taken after rinsing with buffer A. In (C), the edge of the substrate was taken (lower left corner) for contrast purposes. Scale bars: 10 μm.

protein surface coverage of 17 ± 2% was estimated from the fluorescence micrographs by pixel analysis (Figure 3B2). If a buffer with a pH of 4.8 was used, continuous lipid bilayers were obtained as indicated by the blue perylene fluorescence throughout the substrate (Figure 3C1), similar to what has been observed if the membrane was prepared in the presence of 200 mM NaCl. However, while ezrin binding to those membranes was only marginal, a bright green fluorescence was observed upon addition of 0.5 μM AlexaFluor488 labeled ezrin covering the entire membrane (Figure 3C2). Such large ezrin coverage was expected on membranes with 8 mol % PIP2, as quartz crystal microbalance experiments on solid supported hybrid membranes have shown that a PIP2 content of about 7 mol % is sufficient to achieve maximum ezrin coverage.12 14207


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They observed PIP2 domains as a function of pH with enhanced domain formation for pH values larger than the pKa values of the phosphate groups at the 4′ and 5′ position of the inositol ring, which are approximately 6.7 and 7.7.32 This suggests that the deprotonated 4′ and 5′ phosphomonoester groups act as hydrogen bond acceptors. As shown by Liepiņa et al.53 in molecular dynamics simulations, the hydrogen bond acceptors allow the formation of PIP2 clusters in phosphatidylcholine bilayers, bridged by water molecules. In conclusion, these results suggest that PIP2 cluster formation is strongly influenced by hydrogen bond formation and might explain that even in the absence of Ca 2+ an inhomogeneous PIP 2 fluorescence was observed at pH 7.4 as a result of hydrogen bonds leading to PIP2 domains. Hydrogen bond formation and thus PIP2 cluster formation are more likely at pH 7.4 than at low pH value even in the presence of 200 mM NaCl, explaining why a more homogeneous PIP2 fluorescence of the SLBs was observed at pH 4.8 conditions. Lateral Diffusion of PIP2. As all SLBs were eventually analyzed under the same buffer A conditions, the vesicle spreading conditions are apparently the ones that are decisive whether PIP2 clusters visible in the optical microscope are formed. That the formed PIP2 clusters remain stable in the membranes suggests that the immobile fraction and the lateral mobility of the PIP2 molecules are different for the membranes obtained under the different spreading conditions. Thus, we analyzed PIP2 lipid diffusion in SLBs composed of POPC/ PIP2/Bodipy TMR-PIP2/perylene (91:7.5:0.5:1) obtained under the three different buffer conditions by fluorescence recovery after photobleaching (FRAP) experiments. It turned out that the fluorescence obtained from membranes prepared in the presence of Ca2+ was too heterogeneous to be evaluated by FRAP analysis as a result of the additional lipid material attached to the membrane. However, PIP2 lipid diffusion in membranes prepared at pH 4.8 could be readily evaluated by FRAP. A typical time lapse series of a FRAP experiment is shown in Figure 5A (top row, (1a−d)). To quantify the diffusion coefficient and the mobile fraction of Bodipy TMRPIP2, an analysis of the FRAP data according to Jönsson et al.54 was performed. A diffusion coefficient of D = 2.8 ± 0.3 μm2/s was obtained (Figure 5B). Of note, the mobile fraction turned out to be rather small with an average value of 64 ± 3% (Figure 5C). In addition, the fluorescence images show rather defined edges at the bleach spot, which remain clearly visible over time. SLBs prepared in the presence of 200 mM NaCl at pH 7.4 were also subjected to FRAP analysis. Even though the FRAP data could be evaluated, owing to the heterogeneous PIP 2 fluorescence, the error of the diffusion constant as well as that of the mobile fraction was larger with D = 2.0 ± 0.7 μm2/s (Figure 5B) and a mobile fraction of 25 ± 11% (Figure 5C). The results demonstrate that the mobility of PIP2 is significantly reduced in SLBs prepared in the presence of 200 mM NaCl at pH 7.4. As the immobile fraction of PIP2 was very large, independent of the preparation conditions, and the edges of the bleach spot remained clearly visible, we investigated whether the amount of PIP2 in the membrane might be responsible for this observation. Thus, SLBs with only 3 mol % of PIP2 were analyzed by FRAP (Figure 5A, middle row (2a−d)). An average diffusion coefficient of 2.9 ± 0.2 μm2/s was determined (Figure 5B) with a mobile fraction of 52 ± 9% (Figure 5C). This result demonstrates that even at smaller PIP2 concentrations the mobile fraction is only about half of that expected for a fully

Figure 4. Fluorescence images of SLBs composed of POPC/PIP2/ Bodipy TMR-PIP2/perylene (91:7.5:0.5:1). Three different spreading conditions were chosen: (A) buffer A supplemented with 2 mM CaCl2; (B) buffer A, in which 50 mM KCl was replaced with 200 mM NaCl; (C) 20 mM citrate, 50 mM KCl, 0.1 mM EDTA, 0.1 mM NaN3, pH 4.8. All SLBs were rinsed with buffer A after bilayer formation. In the case of Ca2+ promoted spreading, SLBs were additionally rinsed with buffer A containing 10 mM EGTA. (1) Perylene fluorescence of the SLBs. (2) Bodipy TMR-PIP2 fluorescence showing the distribution of the fluorescently labeled PIP2 molecules. Scale bars: 10 μm.

disintegrated the clusters, PIP2 clusters remained rather stable in the SLBs (Figure 4A2). Interestingly, not only the presence of Ca2+ during the spreading process resulted in an inhomogeneous PIP 2 distribution within the membrane but also the presence of 200 mM Na+ at pH 7.4 (Figure 4B2), even though this cation is monovalent and thus not capable of cross-linking PIP2 molecules. Levental et al.51 reported that Ca2+ caused a drop in surface pressure in pure PIP2 monolayers, suggesting a bridging of PIP2 molecules, whereas the addition of Na+ resulted in an increase in surface pressure; i.e., the monolayer got expanded. They attributed this observation to a disruption of a hydrogen bond network between the PIP2 molecules, as pure screening of the negative charges of PIP2 by Na+ could be ruled out. However, these experiments were performed on pure PIP2 monolayers and not on PIP2-doped PC bilayers like in our case. The influence of hydrogen bonds on PIP2 cluster formation in PC vesicles was reported by Redfern et al.52 14208


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Figure 5. Results of fluorescence recovery after photobleaching (FRAP) experiments. (A) Time lapse series of FRAP experiments. Top row (1a−d): fluorescence images of a SLB composed of POPC/PIP2/Bodipy TMR-PIP2/perylene (96:7.5:0.5:1) prepared at pH 4.8 conditions. Middle row (2a− d): fluorescence images of a SLB composed of POPC/PIP2/Bodipy TMR-PIP2/perylene (96:2.5:0.5:1) prepared in buffer A. Bottom row (2a−d): fluorescence images of a SHM composed of POPC/PIP2/Bodipy TMR-PIP2/perylene (96:2.5:0.5:1) on a dodecyltrichlorosilane functionalized silicon substrate prepared in buffer A. Scale bars: 10 μm. (B) Diffusion coefficients and (C) mobile fractions obtained for SLBs and SHMs dependent on the spreading conditions. The error bars are the standard deviation of the mean. SLBs composed of POPC/PIP2/Bodipy TMR-PIP2/perylene (96:7.5:0.5:1) were prepared in 20 mM citrate, 50 mM KCl, 0.1 mM EDTA, 0.1 mM NaN3, pH 4.8 (hatched bar, n = 4) or buffer A, in which 50 mM KCl was replaced with 200 mM NaCl (cross-hatched bar, n = 4), and SLBs composed of POPC/PIP2/Bodipy TMR-PIP2/perylene (96:2.5:0.5:1) were prepared in buffer A (solid bar, n = 10). SHMs were obtained by spreading POPC/PIP2/Bodipy TMR-PIP2/perylene (96:2.5:0.5:1) SUVs in buffer A on dodecyltrichlorosilane-functionalized silicon substrates (solid bars, n = 11). Before the FRAP experiment, SLBs and SHMs were rinsed with buffer A to remove excess lipid material.

results obtained for SLBs, full recovery of the fluorescently labeled PIP2 was found after 16 s for SHMs (Figure 5A, bottom row (3a−d)). A diffusion coefficient for Bodipy TMR-PIP2 of 3.0 ± 0.3 μm2/s (Figure 5B) was obtained, and the mobile fraction was determined to be 98 ± 3% (Figure 5C). This finding supports the notion that PIP2 in the bottom surfaceattached leaflet is almost fully immobile in case of SLBs on hydrophilic silicon substrates. Alteration of PIP2 Lipid Diffusion in SHMs by Ezrin Binding. As the SHMs allowed us to unambiguously observe the diffusion of PIP2 in the top lipid monolayer, we asked the

mobile bilayer. We hypothesized that the PIP2 molecules in the bottom surface-facing leaflet are immobile, while they are mobile in the top layer resulting in a roughly 50% immobile fraction. This idea is supported by the fact that the bleach spot appears to be well-defined even after a longer observation time. To further prove this hypothesis, we prepared supported hybrid membranes (SHMs) on a dodecyltrichlorosilane-functionalized silicon wafer to ensure that only fluorescence from the top monolayer containing fluorescently labeled PIP2 is measured. A rather homogeneous Bodipy TMR-PIP2 fluorescence was observed (Figure 5A, bottom row (3a)). In contrast to the 14209


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reduced the lateral mobility of Bodipy TMR-PIP2 by roughly a factor of 3 (0.7 ± 0.3 μm2/s). This can be explained by the fact that a compact ezrin monolayer on top of the bilayer is formed10 so that the individual ezrin molecules are strongly hindered in their lateral movement, while the underlying still mobile PIP2 molecules in the bilayer reversibly adsorb to and desorb from individual ezrin molecules, which slows down the average macroscopic diffusion coefficient of PIP2. We further investigated the influence of ezrin binding on the distribution of Bodipy TMR-PIP2 within the membrane. Before ezrin binding a homogeneous distribution of the fluorescently labeled PIP2 was observed throughout the entire membrane surface as expected (Figure 6C, 1b). After 2 h of incubation with 0.5 μM AlexaFluor488 labeled ezrin and subsequent rinsing with buffer A, a heterogeneous PIP2 distribution was detected (Figure 6C, 3b). The dark areas in the Bodipy TMRPIP2 fluorescence images cannot be attributed to defects in the lipid monolayer, as the perylene fluorescence was still present in these areas (Figure 6C, 3a). The red fluorescence of the Bodipy TMR-PIP2 colocalized with the AlexaFluor488 labeled ezrin (Figure 6C, 3c). However, this inhomogeneity was not observed throughout the entire membrane area. Also rather homogeneous Bodipy TMR-PIP2 and AlexaFluor488 ezrin fluorescence images were found (Figure 6c, 2b, and 2c). The inhomogeneous PIP2 fluorescence distribution, which colocalized with the ezrin fluorescence, might be interpreted in terms of an ezrin-induced reorganization of PIP2, an aspect that is still controversially discussed in the literature. On the basis of a self-quenching assay with fluorescent analogues of PIP2, Blin et al.14 argued that ezrin binding does not induce PIP2 clustering, whereas binding of MARCKS does. However, the same group observed PIP2 reorganization in GUVs induced by ezrin binding if larger ezrin concentrations up to 15 μM were used.16 They found fluorescently labeled PIP2 colocalized with fluorescently labeled ezrin at GUVs and apparently clustered ezrin-PIP2 complexes in some areas, indicated by enhanced fluorescence. We always observed colocalization of ezrin and PIP2. However, we detected clustering of PIP2 only in certain areas on the membrane, while the PIP2 distribution remained homogeneous in others. We suggest that some membrane areas become depleted in PIP2 upon ezrin binding. This depletion might depend on the local ezrin surface concentration, which is driven by lateral protein−protein interactions. Such ezrin−ezrin interaction on a planar membrane surface has been concluded from atomic force microscopy images showing that an ezrin monolayer is formed emanating from few nucleation points.10

question whether we can observe changes in PIP2 lipid diffusion upon ezrin binding. SHMs composed of POPC/PIP2/Bodipy TMR-PIP2/perylene (96:2.5:0.5:1) were prepared in buffer A and incubated with 0.5 μM AlexaFluor488 labeled ezrin for 2 h. After rinsing with buffer A, the lateral mobility of Bodipy TMRPIP2 was monitored by means of FRAP. After protein binding, a reduced diffusion coefficient of 0.7 ± 0.3 μm2/s was obtained (Figure 6A), which is by a factor of about 3 smaller than that in

Figure 6. PIP2 lipid diffusion and lateral organization of PIP2 as a function of bound ezrin. (A) Diffusion coefficients and (B) mobile fractions of Bodipy TMR-PIP2 within SHMs (POPC/PIP2/Bodipy TMR-PIP2/perylene 96:2.5:0.5:1) in buffer A before (n = 11) and after (n = 15) addition of 0.5 μM AlexaFluor488 labeled ezrin obtained by FRAP analysis. (C) Fluorescence images of a SHM before (1) and after (2/3) incubation with AlexaFluor488 labeled ezrin. Blue: perylene; red: Bodipy TMR-PIP2; green: AlexaFluor488 labeled ezrin. Scale bars: 10 μm.

the absence of bound ezrin, while the mobile fraction of PIP2 only slightly diminished to a value of 89 ± 9% (Figure 6B). No fluorescence recovery was observed if the membrane bound AlexaFluor488 labeled ezrin was bleached, demonstrating that it is immobile on the membrane surface (Supporting Information, Figure S4). Golebiewska et al.55 microinjected Bodipy TMR-PIP2 into rat cells and determined the diffusion constants. They determined an average diffusion coefficient for Bodipy TMR-PIP2 of 0.9 ± 0.2 μm2/s in the unperturbed plasma membrane, while they found a value of 2.5 ± 0.8 μm2/s in blebs formed on the plasma membrane. Blebs are characterized by poor coupling to the cytoskeleton, thus resembling the situation of an artificial phospholipid bilayer better. An average diffusion coefficient of 3.3 ± 0.8 μm2/s for Bodipy TMR-PIP2 was also reported in GUVs.56 The obtained diffusion coefficient of 3.0 ± 0.3 μm2/s in our study is hence in the same range as reported diffusion coefficients of Bodipy TMR-PIP2 in free-standing artificial and natural membranes. Binding of the protein ezrin significantly

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CONCLUSIONS The formation of continuous lipid bilayers composed of POPC and doped with 8 mol % PIP2 on hydrophilic silicon substrates can only be achieved under particular buffer conditions. Only a low pH value of 4.8, which results in a reduced net negative charge of the PIP2 molecules, allowed the reproducible spreading of small unilamellar vesicles to form planar lipid bilayers so that a PIP2 binding protein such as ezrin binds to the PIP2 receptor lipids with large surface coverage. The tendency of PIP2 to form clusters depends on the buffer conditions. These clusters together with the altered diffusive behavior of the PIP2 molecules as a result of the buffer conditions, and the underlying substrate can strongly influence the outcome of quantitative measurements on PIP2 binding proteins using supported membranes in conjunction with surface-sensitive methods. 14210


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Preparation of Solid Supported Membranes. For fluorescence analysis and fluorescence recovery after photobleaching (FRAP) experiments either supported lipid bilayers (SLBs) or supported hybrid membranes (SHMs) were prepared (Scheme 1). A silicon substrate (100 nm SiO2 layer, 0.8 × 2.0 cm2) was rinsed thoroughly with isopropanol and water. An aqueous solution of NH3 and H2O2 (H2O/NH3/H2O2, 5:1:1) was used to turn the silicon substrate’s surface hydrophilic and remove organic contaminations (20 min at 70 °C). Additional hydrophilization was achieved by oxygen plasma treatment for 2 min. To form SLBs, the hydrophilized substrate was directly treated with freshly prepared SUV suspension (0.2 mg/mL) and incubated for 2 h. Subsequent rinsing with buffer A removed remaining lipid material from solution. For the preparation of SHMs, the hydrophilized silicon substrates were silanized with dodecyltrichlorosilane as described.58 The substrate was immersed in dry toluene containing dodecyltrichlorosilane (2% (v/v)) under vacuum for 15 min, thoroughly rinsed with dry toluene, and left overnight under vacuum at 65 °C. Toluene p.a. was dried over 4 Å molecular sieves. After the silanization, the dodecyltrichlorosilane functionalized substrate was incubated with the SUV suspension as described above. Reflectometric Interference Spectroscopy (RIfS). RIfS was employed to monitor the formation of SLBs and subsequent protein binding in a time-resolved manner.59 The experimental setup used in this work has been described elsewhere.60 Briefly, RIfS experiments were performed using a NanoCalc-2000 vis/NIR spectrometer (Ocean Optics, Dunedin, FL). Silicon wafers with a 5 μm think SiO2 layer were used to generate a sufficiently thick film for interference fringes. Spectra were recorded every 2 s, and data were evaluated using a MATLAB tool. Fluorescence Microscopy and Fluorescence Recovery after Photobleaching (FRAP). Fluorescence imaging and FRAP experiments were performed on a LSM 710 confocal fluorescence laser scanning microscope (Carl Zeiss, Jena, Germany). The microscope was equipped with a water immersion 63× objective with a numerical aperture of NA = 1 (W Plan Achromat, Carl Zeiss, Jena, Germany) and operated with the software Zen 2008. An Ar laser was used for the excitation of AlexaFluor488-labeled ezrin (λex = 488 nm, λem = 495− 575 nm), the perylene fluorescence was excited at λex = 405 nm by a diode laser and detected at λem = 410−480 nm, and a He−Ne laser was chosen for the excitation of Bodipy-TMR-PIP2 (λex = 561 nm, λem = 574−619 nm).12 The Hankel transform method was used to analyze the acquired FRAP data.54

EXPERIMENTAL SECTION

Materials. L-α-Phosphatidylinositol-4,5-bisphosphate (PIP2, purified from porcine brain with a fatty acid composition primarily composed of 18:0, 18:1, and 20:4 acyl chains) and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) were obtained from Avanti Polar Lipids (Alabaster, AL). The fatty acid fluorescently labeled PIP2 analogue Bodipy TMR-PIP2 (C16) was from Echelon Biosciences (Salt Lake City, UT). The fluorophores perylene, βBodipy 500/510 C12-HPC, and AlexaFluor488 C5-maleimide were obtained from Life Technologies (Darmstadt, Germany). Dodecyltrichlorosilane and isopropyl-β-D-thiogalactopyranoside were from Sigma-Aldrich (Steinheim, Germany). Silicon wafers were purchased from Silicon Materials (Kaufering, Germany). Toluene p.a. was from VWR (Darmstadt, Germany), and 4 Å molecular sieves were obtained from Carl Roth (Karlsruhe, Germany). Protein Purification. Ezrin was obtained by recombinant expression in Escherichia coli cells (strain BL21(DE3)pLysS) and purified as described.57 Briefly, transformed E. coli cells containing the bacterial expression vector pET28a+ (Novagen, Madison, WI) encoding ezrin with an N-terminal histidine tag were grown to an OD600 of 0.6. Protein expression was induced by adding 1 mM isopropyl-β-D-thiogalactopyranoside. After 3 h, the cells were harvested by centrifugation (4000g, 20 min, 4 °C), and the pellet was resuspended in lysis buffer (40 mM HEPES, 20 mM imidazole, 300 mM NaCl, 1 mM EDTA, 10 mM mercaptoethanol, protease inhibitor cocktail (Complete; Roche Diagnostics, Basel, Switzerland), pH 7.4). To complete lysis, the suspension was sonicated on ice. The bacterial lysate was clarified by centrifugation (100000g, 1 h, 4 °C), and the supernatant was applied to a Ni-nitrilotriacetic acid (Ni-NTA) agarose column (Qiagen, Hilden, Germany) equilibrated with lysis buffer. The column was washed twice with lysis buffer supplemented with 30 mM imidazole, pH 7.4 and 50 mM imidazole, pH 7.4, respectively. Ezrin was eluted using buffer A (20 mM Tris/HCl, 50 mM KCl, 0.1 mM EDTA, 0.1 mM NaN3, pH 7.4) containing 250 mM imidazole, dialyzed against imidazole-free buffer A, and stored at 4 °C until use. Protein concentration was determined by UV/vis spectroscopy using an extinction coefficient of ε280 = 66 900 M−1 cm−1. Protein Labeling. Ezrin labeling was carried out as described.12 Ezrin was dissolved in buffer A at 20 μM and mixed with dithiothreitol (DTT) in a 10-fold molar excess. The protein solution was dialyzed overnight against DTT-free, degassed buffer A. AlexaFluor488 C5maleimide dissolved in degassed, ultrapure water at a molar ratio of 20:1 (AlexaFluor488 C5-maleimide:ezrin) was added dropwise to the protein solution, and the reaction mixture was stirred overnight. The addition of 2 mM mercaptoethanol quenched the reaction. Labeled protein was separated from free dye by size exclusion chromatography (Sephadex-NAP 25; GE Healthcare, Piscataway, NJ). All steps were carried out at 4 °C. The labeling efficiency was evaluated by UV/vis spectroscopy (ε493 = 71 900 M−1 cm−1) yielding a dye-to-protein ratio of 1:1. Vesicle Preparation. Stock solutions of the respective lipids were prepared in chloroform at concentrations ranging from 0.5 to 10 mg/ mL except for PIP2, which was dissolved in a mixture of chloroform/ methanol/water (20:9:1) at 1 mg/mL. Lipid stock solutions (0.4−0.5 mg of total lipid) were mixed in a test tube preloaded with 200 μL of chloroform at the desired molar ratio. Fluorophores were added as indicated. The organic solvent was evaporated with a gentle stream of nitrogen at a temperature above the lipid gel−fluid phase transition. To remove residual solvent, the lipid film was further dried under vacuum for 3 h at the same temperature. Lipid films were stored at 4 °C until use. Small unilamellar vesicles (SUVs) were prepared by sonication. A lipid film was rehydrated by adding 0.5−1.0 mL of buffer solution (composition according to chosen spreading conditions), incubated for 20 min, and vortexed for 3 × 30 s periods with 5 min rest in between. The suspension of multilamellar vesicles was transferred to an Eppendorf cup and sonicated for 2 × 15 min using an ultrasonic homogenizer (Sonopuls HD2070, resonator cup, Bandelin; Berlin, Germany).

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ASSOCIATED CONTENT

S Supporting Information *

Kinetics of SUV adsorption and spreading containing different PIP2 concentrations on hydrophilic silicon substrates; fluorescence images of a SLB formed upon Ca2+-induced spreading of POPC/PIP2 SUVs; RIfS experiment showing the influence of Ca2+ incubation of ezrin binding; FRAP experiment showing the immobility of membrane bound ezrin. 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.). Author Contributions †

J.A.B. and C.K. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to M. Stephan for providing the MATLAB tool used for RIfS experiments and to J. Heise and L. Kahmann for performing RIfS experiments. We thank the DFG for financial support (STE 884/11-1). 14211


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Solid Supported Membranes Doped with PIP2: Influence of Ionic

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