A Microbead Supported Membrane-Based Fluorescence Imaging

Jun 4, 2016 - Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore. ‡. Department of Chemistry, University of Cal...
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A Microbead Supported Membrane-Based Fluorescence Imaging Assay Reveals Intermembrane Receptor−Ligand Complex Dimension with Nanometer Precision Kabir H. Biswas*,† and Jay T. Groves*,†,‡ †

Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore Department of Chemistry, University of California, Berkeley, California 94720, United States

‡

ABSTRACT: Receptor−ligand complexes spanning a cell− cell interface inevitably establish a preferred intermembrane spacing based on the molecular dimensions and orientation of the complexes. This couples molecular binding events to membrane mechanics and large-scale spatial organization of receptors on the cell surface. Here, we describe a straightforward, epi-fluorescence-based method to precisely determine intermembrane receptor−ligand dimension at adhesions established by receptor−ligand binding between apposed membranes in vitro. Adhesions were reconstituted between planar and silica microbead supported membranes via specific interaction between cognate receptor/ligand pairs (EphA2/EphrinA1 and E-cadherin/anti-E-cadherin antibody). Epifluorescence imaging of the ligand enrichment zone in the supported membrane beneath the adhering microbead, combined with a simple geometrical interpretation, proves sufficient to estimate intermembrane receptor−ligand dimension with better than 1 nm precision. An advantage of this assay is that no specialized equipment or imaging methods are required.

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INTRODUCTION A large number of cellular proteins are expressed as cell membrane receptors that serve to integrate signals from the cellular microenvironment. A subset of these receptors interacts with ligands on another cell surface, thus establishing a cell−cell junction. These include the Eph family of receptor tyrosine kinases and their ephrin family of ligands,1−6 cadherin family of adhesion molecules,7−10 T-cell receptor (TCR) and peptidemajor histocompatibility complex (pMHC) along with a wide variety of other immune modulator proteins,11,12 immunoglobulin family of adhesion molecules,13,14 or Notch and Delta/ Serrate/LAG-2 (DSL) family of proteins.15,16 An interesting feature of these molecules is the variation in their dimension, and it has been suggested that such physical differences drives their segregation into discrete zones in a multicomponent adhesion (Figure 1a). A classical example of this is observed in the formation of the immunological synapse during T-cell activation. In these intercellular junctions, the TCR/pMHC complex localizes to the central zone while the lymphocyte function-associated antigen-1 (LFA-1)/Intercellular Adhesion Molecule -1 (ICAM-1) complex localize peripherally in the immunological synapse.11,17,18 Further, artificial perturbations of a receptor/ligand complex have been shown to give rise to a dimension-based segregation of the mutant from the wild type complex.18−22 Insights obtained from these studies are being utilized to develop cancer therapeutic strategies using bispecific antibodies and related molecules that recognize a specific cell surface protein on tumor and/or immune cells to induce a therapeutically beneficial immune response.23,24 © 2016 American Chemical Society

A number of methods are available to determine the intermembrane receptor−ligand spacing in adhesions. These include fluorescence resonance energy transfer (FRET)-based detection of membrane localized fluorophore separation25 or fluorescence lifetime imaging microscopy (FLIM) of protein structures on solid surfaces.26 Both of these techniques are based on nonradiative energy transfer and are often too short ranged (∼5 nm) to measure intercellular complexes, which can span tens of nanometers. Many interferometric methods overcome this limitation, including reflection interference contrast (RICM), fluorescence interference contrast microscopy (FLIC),27,28 or scanning angle interference microscopy (SAIM).29 Other methods that can determine intermembrane separation at high-resolution include differential evanescence nanometry,30 a modified, two-wavelength total internal reflection fluorescence (TIRF)-based method31 or direct or modified forms of surface force apparatus measurements.32−37 While all of these methods can provide nanometer scale resolution over distances relevant to cell adhesions, they also require specialized instrumentation or complex data analysis, making them inaccessible to a wider community of researchers. Here, we describe a precise yet simple method to estimate the intermembrane receptor/ligand dimension in adhesions formed by interaction between cognate receptor/ligand pair. The method is based on in vitro reconstitution of adhesion between a planar and a silica microbead-supported membrane. Received: April 11, 2016 Revised: May 24, 2016 Published: June 4, 2016 6775

DOI: 10.1021/acs.langmuir.6b01377 Langmuir 2016, 32, 6775−6780

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Use of membrane preserves the native-like display of the proteins and allows assembly of high density adhesions enabled by the inherent mobility of proteins attached to lipid molecules in a membrane. Membrane-coated microbeads have been successfully utilized for applications such as detection of protein−protein interactions38,39 and studying the effect of electrostatic charge on the interaction between colloidal particles and membranes.40,41 We take advantage of the geometric constraint imposed by the spherical shape of the microbead to allow assembly of spatially defined adhesions. Adhesions are observed as a zone of enrichment of the fluorescent ligand on the planar glass-supported membrane beneath the microbead by epi-fluorescence imaging. Measured size of an adhesion is then geometrically interpolated to calculate the average intermembrane receptor/ligand complex dimension with less than 1 nm precision. The method can be easily translated to other receptor/ligand pairs and may be useful in testing the effect of genetic mutations and sizedependent segregation of complexes at multicomponent adhesions or determining changes in the intermembrane separation under different conditions that causes a change in the receptor/ligand configuration or orientation.

Figure 1. Schematic representation of an adhesion formed by membrane-associated receptor/ligand complexes of two different sizes. (a) A schematic representation of two cells adhering to each via interaction between two structurally distinct pairs of receptor/ ligand complexes. A zoomed-in view of the multicomponent adhesion showing segregation of receptor/ligand complexes based on their lengths. (b) A schematic representation of the adhesion reconstituted in vitro. Receptor/ligand complexes form adhesion enrichment based on their molecular length and binding orientation; that is, a short complex with length S′ will form a smaller adhesion of radius r′ while a long complex with length S′′ will form a larger adhesion of radius r″.

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EXPERIMENTAL SECTION

Supported membranes were deposited as described previously.42 1,2Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was mixed with either 4 mol % of 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] nickel salt (Ni−NTA-DOGS) or 0.2 mol

Figure 2. Spatially defined adhesion formed by receptor−ligand complexes between planar and microbead supported membranes. (a,d,g) Schematic representations of GFP (a), EphA2/EphrinA1−EYFP (d) and E-cadherin/anti-E-cadherin antibody (g) adhesion. (b,e,h) Bright-field, RICM, and epi-fluorescence images of a control microbead without any protein (b), or microbeads functionalized with either EphA2 (e) or E-cadherin (h) on planar supported membranes functionalized with GFP (b), EphrinA1 (e) or anti-E-cadherin antibody (h). Note that EphrinA1 and anti-E-cadherin antibody functionalized membranes show enrichment of the protein on the planar bilayer while no enrichment of EGFP is seen with control microbead. Insets in (e,h) show zoomed-in view of a 2 μm by 2 μm area containing an adhesion. (c,f,i) Plots of RICM and fluorescence intensity obtained from line scan analysis of the control EGFP functionalized membrane (c) or membranes functionalized with either EphrinA1−EYFP (f) or anti-E-cadherin antibody (i). Note the fluorescence intensity peak in (f,i) matches with the position of maximum intensity loss in the RICM image. Scale bar: 5 μm. 6776

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Langmuir % of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (16:0 biotinyl-Cap-PE) in chloroform. All lipids were purchased from Avanti Polar Lipids (Alabaster, Alabama, U.S.A.). A thin lipid film was deposited on a round bottle flask by rotary vacuum. Lipids were then resuspended in deionized H2O to a final concentration of 0.5 mg/ mL and sonicated (Sonics Vibra Cell, Sonics & Material, Newton, Connecticut, U.S.A.) to generate small unilamellar vesicles (SUVs). Vesicle suspensions were centrifuged at 20 000g and 4 °C for 3 h, and supernatants were collected. All lipid vesicles were stored at 4 °C until further use. Glass coverslips were cleaned sequentially by sonication in a 1:1 mixture of isopropanol and water for 30 min, overnight incubation in 50% H2SO4 and UV treatment in an enclosed UV ozone generator (UV/Ozone ProCleaner Plus, Bioforce Nanosciences, Ames, Iowa, U.S.A.) for 30 min. Glass coverslips were washed thoroughly with deionized H2O after every step and finally dried under an N2 stream before bilayer deposition. Cleaned silica microbeads with an average diameter of 5.2 μm (Bang Laboratories Inc., Fishers, Indiana, U.S.A.) were utilized for all experiments presented here. Because of the resolution limit of an optical microscope, microbeads of appropriate size should be used to minimize error in the determination of the adhesion size. Membranes were assembled on both coverslips and microbeads by incubating a 1:1 mixture of lipid vesicles and 2× Trisbuffered saline (TBS) for 5 min. Coverslips were then each assembled into an Attofluor Cell Chambers purchased from Life Technologies (Carlsbad, California, U.S.A.). His6-EGFP (GFP) and EphrinA1−EYFP-His1043 were purified from bacteria using Ni−NTA beads. C-terminal His6-tagged extracellular domain constructs of EphA2 and E-cadherin were purchased from Sino Biologicals (Beijing, China). Cascade blue conjugated neutravidin was purchased from Lifetechnologies (Carlsbad, California, U.S.A.) and biotinylated monoclonal anti-E-cadherin antibody was purchased from R&D Systems (Minneapolis, Minnesota, U.S.A.). All membranes were first blocked by incubating with 0.01% bovine serum albumin (BSA) in TBS for 30 min and washed thrice after the incubation with TBS. Membranes were then functionalized with specific proteins by incubating an appropriate amount of the protein in TBS for 90 min and washed thrice after the incubation with TBS. Calcium was included at a concentration of 1 mM in case of E-cadherin. Biotincontaining membranes were preincubated with neutravidin for 30 min. Adhesions were reconstituted by the interaction of either EphA2 and EphrinA1 or E-cadherin and anti-E-cadherin antibody functionalized membranes for 1 h. A GFP functionalized membrane was used as a negative control. Bright-field, epi-fluorescence, and RICM images of the adhesions were acquired using an Eclipse Ti inverted microscope (Nikon, Minato-ku, Tokyo, Japan) equipped with an Evolve EMCCD camera (Photometrics, Tucson, Arizona, U.S.A.). Images were collected with MetaMorph (Molecular Devices, Sunnyvale, California, U.S.A.) and analyzed with Fiji44. Radial intensity profiles of the adhesions were generated using the Radial Profile ImageJ plugin and cubic spline fitting of the radial intensity profiles was performed using Matlab (The MathWorks, Inc., Natick, Massachusetts, U.S.A.). All graphs were generated and frequency histograms were fitted using GraphPad Prism (GraphPad Software, San Diego California, U.S.A.).

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distance will be determined by the length and orientation of the receptor/ligand complex. We tested the specificity of the experimental system by incubating microbeads without any receptor protein while the planar membrane displayed GFP protein via Ni−NTA interaction42 (Figure 2a). Although RICM imaging showed a close juxtaposition of the microbead with the planar membrane,40,41 no enrichment of GFP on the planar bilayer was observed (Figure 2b,c), suggesting that the microbead rests on the planar surface due to gravity without any adhesion. We then used a natural receptor−ligand pair, EphA2−EphrinA1,1,5,45,46 to establish adhesion between the two membranes. Both EphA2 and ephrinA1 are expressed on the cell membrane and interaction between them has been implicated in physical force sensing in breast cancer cells.47 The extracellular domain of EphA2 contains the ligand-binding domain that binds EphrinA1 with high affinity. Molecular binding of EphrinA1 to EphA2 results in the formation of microclusters that mediate EphA2 signaling.43,47−49 Microbead-supported membrane was functionalized with EphA2 while the planar-supported membrane was functionalized with fluorescent EphrinA1−EYFP. Both proteins were anchored to the respective membranes through Ni−NTA interaction.50 Incubation of EphA2 functionalized microbeads with EphrinA1−EYFP functionalized planar supported membrane (Figure 2d) resulted in a clear enrichment of EphrinA1−EYFP at the site of contact between the two membranes (Figure 2e,f), indicating the formation of an EphA2/EphrinA1−EYFP adhesion. Importantly, fluorescence intensity profiling across the adhesion showed no depletion of the EphrinA1−EYFP molecule in the supported bilayer region surrounding the adhesion (Figure 2f), indicating the absence of any detectable immobile fraction of the ligand on the bilayer. In order to further test the utility of the method developed here, we reconstituted an artificial adhesion consisting of Ecadherin and an antibody against the extracellular domain of Ecadherin (Figure 2g). E-cadherin is a type I, calcium-dependent cell adhesion molecule that is the principle component of cell− cell junctions in epithelial cells.42,51 The biotinylated anti-Ecadherin antibody was functionalized to the planar supported membrane via biotin-neutravidin interaction while E-cadherin was functionalized to the microbead-supported membrane via Ni−NTA interaction. Fluorescent label on neutravidin allowed imaging of adhesions in this case. As seen with EphA2EphrinA1, incubation of E-cadherin functionalized microbeads with the anti-E-cadherin antibody functionalized planar membrane resulted in an enrichment of the antibody specifically at the site of microbead contact (Figure 2h,i), indicating the formation of an artificial adhesion between Ecadherin and anti-E-cadherin antibody. Having reconstituted minimal adhesions, we went ahead to determine the intermembrane receptor/ligand complex dimension in these adhesions (Figure 3a,b,c). Epi-fluorescence images of adhesions were analyzed for detection of adhesions followed by the generation of a radial intensity profile for each adhesion (Figure 3c). The profiles were fitted to a cubic spline to determine half radial distance of the adhesion, s. This relates to the average receptor/ligand length, assuming that the intermembrane separation varies linearly in the small adhering surface area of the microbead relative to the total surface area of the microbead (Figure 3b). Importantly, the determination of s from the relative fluorescence intensity profile renders it insensitive to the presence of immobile proteins on the bilayer, photobleaching and imaging light intensity. The intermem-

RESULTS AND DISCUSSION

To determine the intermembrane receptor/ligand complex dimension, we reconstituted adhesions between a planar- and a microbead-supported membrane displaying a ligand and a receptor, respectively. The key to this method is the use of spherical microbeads due to which the separation between the two membranes increases with increasing lateral distance from the contact point (which is the center of an adhesion). Therefore, a receptor−ligand pair can physically interact with each other for up to a certain finite distance from the center of the adhesion (Figure 1b). For a given microbead size, this 6777

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few dozen are utilized here. The standard error, δS , in the determination of S from a single microbead image is given by δS =

⎛ ∂S ⎞2 ⎛ ∂S ⎞2 2 2 ⎜ ⎟ (δs) + ⎜ ⎟ (δR ) ⎝ ∂s ⎠ ⎝ ∂R ⎠

where δs and δR represent the standard error in the determination of the size of the adhesion (roughly equal to the image pixel size of 160 nm) and the variation in the actual size of the microbeads (∼130 nm).52 On the basis of this analysis, the standard error for each individual measurement of S is ∼25 nm, which is comparable to the observed variation in the E-cadherin/anti-E-cadherin antibody complex and somewhat broader than that observed for the EphA2/EphrinA1− EYFP complex. In both cases, however, the SEM from N individual microbead measurements scales with 1/√N, leading to the high precision achieved. The structure of the EphA2 extracellular domain in complex with EphrinA5 (PDB: 2X11)4,5 shows a length of 17 nm while the diameter of EYFP molecule (N- and C-termini of the barrel shaped structure ends on the same side, hence the use of diameter) is 2 nm (Figure 3d). Additionally, the His-tag and the linker between EphrinA1 and EYFP will contribute to the dimension. Thus, a mean intermembrane receptor/ligand length of 20 ± 0.1 nm for EphA2/EphrinA1−EYFP complex obtained from the fluorescence data (Figure 3e) appears to be in good agreement with structural data, indicating that the method developed here provides precise determination of intermembrane receptor-ligand complex dimension. Unlike the EphA2/EphrinA1−EYFP complex, neither the structure for E-cadherin/anti-E-cadherin antibody complex nor the exact binding epitope of the antibody is known (the monoclonal anti-E-cadherin antibody was raised against the recombinant, full-length extracellular domain of human Ecadherin spanning residues Asp155-Ile707). The approximate end-to-end length of an E-cadherin molecule is 20 nm (PDB: 3Q2V)8 while the length of an antibody molecule is 12 nm (PDB: 1IGT)53 (Figure 3f). Additionally, neutravidin (PDB: 4JO6)54 will add ∼5 nm to the length of the complex (Figure 3f). Thus, a mean intermembrane receptor−ligand complex dimension of 28 ± 0.8 nm for E-cadherin/anti-E-cadherin antibody complex suggests that the antibody binds close to the second cadherin domain (EC2) of E-cadherin.

Figure 3. Determination of the intermembrane receptor−ligand complex dimension. (a) Schematic illustration of an adhesion formed between a planar and microbead supported membrane and its geometry. The radius of the microbead and the thickness of the membrane are represented together as R, intermembrane receptor− ligand complex length at a radial distance of half maximal fluorescence intensity as S and radial distance at half-maximum intensity as s. (b) Zoomed-in view of the geometry of the adhesion highlighting the right angled triangle with sides R, R + S , and s in green. (c) A representative radial profile of an adhesion showing s obtained from cubic spline fitting of the original fluorescence intensity data. (d,f) Schematic illustration of the molecular interaction between EphA2/EphrinA1− EYFP (d) and E-cadherin/anti-E-cadherin antibody (f). Lengths of each molecule or complex obtained from crystallographic data are also shown. (e,g) Frequency histogram of intermembrane receptor/ligand complex dimension for EphA2/EphrinA1−EYFP (N = 61) (e) and Ecadherin/anti-E-cadherin antibody (N = 85) (g) adhesions. Data was fit to a Gaussian, and mean ± SEM (precision) obtained from the fit are shown.

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CONCLUSION We present a simple yet precise method to determine intermembrane receptor/ligand complex dimension in adhesions formed by membrane-associated proteins. Spatially defined interaction of a receptor with its cognate ligand presented on microbead and planar supported membranes, respectively, allows assembly of adhesion between the two membranes. A straightforward geometric interpretation of the adhesion size provides intermembrane separation or the receptor/ligand complex dimension. It is a general method that can be applied to any receptor−ligand pair that can be purified, fluorescently labeled and coupled to a membrane.

brane receptor/ligand complex length, S , was then calculated using the geometric relation: S = R2 + s 2 − R , where R = 2606 nm [mean radius of the microbead (2600 nm) + a layer of water (1 nm) + membrane thickness (5 nm)]. Using this equation, we calculated the mean intermembrane receptor/ ligand complex dimension and its standard error of mean (SEM) for both EphA2/EphrinA1−EYFP and E-cadherin/antiE-cadherin antibody by fitting the frequency histogram to a Gaussian. This analysis revealed a mean intermembrane receptor/ligand complex dimension of 20 ± 0.1 nm for the EphA2/EphrinA1−EYFP and 28 ± 0.8 nm for E-cadherin/antiE-cadherin antibody adhesions (Figure 3e,g). The extremely high precision afforded by this methodology fundamentally stems from the fact that many individual adhesion zones are imaged. In fact, it is possible to image up to thousands of microbeads in a single frame, although only a

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.T.G.). *E-mail: [email protected] (K.H.B.). Notes

The authors declare no competing financial interest. 6778

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ACKNOWLEDGMENTS We would like to acknowledge the support provided by the National Research Foundation (NRF) through the Mechanobiology Institute, National University of Singapore and NRF CRP Grant CRP001-084 (J.T.G). We would also like to thank Chen Zhongwen for providing a biotin lipid containing vesicle preparation and Oh Hui Ting for help with data analysis.

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DOI: 10.1021/acs.langmuir.6b01377 Langmuir 2016, 32, 6775−6780

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DOI: 10.1021/acs.langmuir.6b01377 Langmuir 2016, 32, 6775−6780