Article pubs.acs.org/Langmuir
Formation and Characterization of Supported Lipid Bilayers Containing Phosphatidylinositol-4,5-bisphosphate and Cholesterol as Functional Surfaces Patrick Drücker,† David Grill,‡ Volker Gerke,‡ and Hans-Joachim Galla*,† †
Institute of Biochemistry and ‡Institute of Medical Biochemistry, ZMBE, University of Münster, D-48149 Münster, Germany S Supporting Information *
ABSTRACT: Solid-supported lipid bilayers (SLBs) mimicking a biological membrane are commonly used to investigate lipid− lipid or lipid−protein interactions. Simple binary or ternary lipid systems are well established, whereas more complex model membranes containing biologically important signaling lipids such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and cholesterol have not been extensively described yet. Here we report the generation of such bilayers and their relevant biophysical properties and in particular the accessibility of PI(4,5)P2 for protein binding. Ternary mixtures of POPC with 20% cholesterol and either 3 or 5 mol % dioleoylphosphatidylinositol-4,5-bisphosphate were probed by employing the quartz crystal microbalance and atomic force microscopy. We show that these mixtures form homogeneous solid-supported bilayers that exhibit no intrinsic phase separation and are characterized by long-term stability (>8 h). Bilayers were formed in a pH-dependent manner and were characterized by the accessibility of PI(4,5)P2 on the SLB surface as shown by the interaction with the PI(4,5)P2 binding domain of the cortical membrane-cytoskeleton linker protein ezrin. A time-dependent reduction of PI(4,5)P2 levels in the upper leaflet of SLBs was observed, which could be effectively inhibited by the incorporation of a negatively charged lipid such as phosphatidylserine. Furthermore, quartz crystal microbalance measurements revealed that cholesterol affects bilayer adsorption to the solid support.
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INTRODUCTION The reproduction of a cellular membrane in a biophysical model system is of major importance in studying the lipid phase behavior and lipid−protein interactions in a quantitative manner. Examples of such applications include the analysis of protein−lipid interactions leading to phase separation in initially homogeneous lipid bilayers and the formation of lipid microdomains, sometimes called rafts.1−5 Biophysical parameters obtained in these experiments are apparent dissociation constants or cooperativity measures of binding reactions. The mammalian plasma membrane is rich in phosphatidylcholines (PC), phosphatidylserines (PS), and phosphatidylinositols on the inner cytoplasmatic leaflet,6,7 with the negatively charged lipid PS comprising about 15−20%. Cholesterol is another important component in regulating membrane structure and lipid organization and can be efficiently sequestrated into membrane microdomains.5,6,8 In vertebrate membranes, the typical levels of cholesterol vary between 30 and 40%.9 However, limiting the amount to 20% has several advantages in model membrane systems. For instance, high levels of cholesterol may enhance transition temperatures or induce the phase separation of lipid mixtures.10 Fluid homogeneously mixed membranes, however, are a prerequisite to the study of protein-induced microdomains. © 2014 American Chemical Society
The presence of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and cholesterol in model membranes is important to the investigation of biologically relevant models. PI(4,5)P2 is often present in raftlike microdomains where it plays a role in signal transduction, for instance, by recruiting and concentrating signaling complexes.11,12 Previous work covers a plethora of lipid mixtures including mixtures of either PI(4,5)P2 and PC13,14 or cholesterol and PS used as solid-supported bilayers (SLBs). However, the combination of PI(4,5)P 2 and cholesterol as found in natural membranes has not yet been considered in detail.15−17 Here we characterize PI(4,5)P2 and cholesterol containing ternary and complex membrane systems with respect to their ability to form homogeneous solidsupported bilayers (SLB) with the receptor lipid PI(4,5)P2 being accessible on the receptive membrane surface. The choice of the supported model membrane is also crucial in many applications. We investigated lipid mixtures with moderate PI(4,5)P2 levels that resemble to some extent the concentration of ∼1% being present in the eukaryotic plasma membranes.12,18 Moderately enhanced levels of 3−5% PI(4,5)Received: August 11, 2014 Revised: November 20, 2014 Published: November 21, 2014 14877
dx.doi.org/10.1021/la503203a | Langmuir 2014, 30, 14877−14886
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nm pore size polycarbonate membrane (Avestin Liposofast, Ottawa, Canada). Preparation of Solid-Supported Bilayers. Silicon surfaces were cleaned and hydrophilized by 10 min of UV/ozone treatment, 30 min of rinsing in 2% (w/v) SDS (SERVA, Heidelberg, Germany), 10 min of UV/ozone treatment, and finally 3 min of O2-plasma treatment (Harrick Plasma, Ithaca, NY). Subsequently, surfaces were rinsed with ultrapure water and dried in a stream of nitrogen. Small unilamellar vesicles (0.1−0.2 mg/mL, 50 nm) were incubated at T > Tm,lipid on the support. To prepare the supported hybrid layer membrane, a modified protocol was used.14 Briefly, dry toluene/dry trichlorododecylsilane (2% v/v) was incubated on hydrophilized substrates at 50 °C in vacuum (∼100 mbar) for 15 min, rinsed with dry toluene, dried in a stream of nitrogen, and left overnight in high vacuum at 60 °C. The quality of the hydrophobized surface was verified using contact angle measurements (dynamic sessile drop method) that typically yielded a contact angle of Θ ≈ 120−125° (Krüss DSA 100, Krüss GmbH, Hamburg, Germany). The hydrophobic surface was then incubated with the SUV suspension as described before for SLB preparation. Following membrane formation, the citrate buffer was exchanged with HBS pH 7.4 buffer by gentle washing. QCM Measurements. QCM measurements were performed on a Q-Sense E4 QCM-D (Q-Sense, Gothenburg, Sweden) at 20 °C. The flow cells were connected to a peristaltic pump (Ismatec IPC, Glattbrugg, Switzerland) employing a flow rate of 80.4 μL/min. Frequency and dissipation shifts of the seventh overtone resonance frequency of the quartz sensor (QSX 303, 50 nm SiO2, 5 MHz) were considered for evaluation. AFM Measurements. AFM measurements were performed using a NanoWizard 3 BioScience AFM (JPK Instruments AG, Berlin, Germany) on a Zeiss Axio Observer D.1 (Carl Zeiss AG, Oberkochen, Germany). Data analysis employed the JPK data processing software and Gwyddion. Intermittent contact measurements were made at a 1 Hz line rate using an MSCT cantilever with a nominal spring constant of 0.03−0.1 N/m and a silicon nitride tip. For calibration, thermal noise measurements were used. The force curve tip velocity was 0.5 μm/s. Young’s moduli were calculated using JPK’s data processing software and a Hertz model, spherical indenter, tip radius of ∼10 nm, and Poisson ratio of 0.5. The model is appropriate for measurements with low indentation. Applying a Hertz model for a parabolic tip leads to a minimal change in the result of
