Probing the Interface Structure of Adhering Cells by Contrast Variation

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Biological and Environmental Phenomena at the Interface

Probing the interface structure of adhering cells by contrast variation neutron reflectometry Philip Böhm, Alexandros Koutsioubas, Jean-François Moulin, Joachim O. Rädler, Erich Sackmann, and Bert Nickel Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02228 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018

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Probing the interface structure of adhering cells by contrast variation neutron reflectometry Philip Böhm1,2, Alexandros Koutsioubas3, Jean-François Moulin4, Joachim O. Rädler1,2, Erich Sackmann5, Bert Nickel1,2

1

Fakultät für Physik and Center for NanoScience, Ludwig-Maximilians-Universität, Geschwister-Scholl-Platz 1, 80539 München, Germany 2

3

Nanosystems Initiative Munich, Schellingstraße 4, 80799 München, Germany

Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Jülich GmbH, Lichtenbergstr. 1, 85748 Garching, Germany

4

Helmholtz-Zentrum Geesthacht, Zentrum für Material und Küstenforschung,

Außenstelle am MLZ in Garching bei München, Lichtenbergstraße 1, 85748 Garching, Germany 5

Physikdepartment E22, Technische Universität München, James-Franck-Str.1, 85748 Garching, Germany

KEYWORDS: Neutron Reflectometry, Cell Adhesion, Membrane, Contrast Variation ACS Paragon Plus Environment

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Abstract

Cellular adhesion is a central element in tissue mechanics, biological cell-cell signaling, and cell motility. In this context, the cell-substrate distance has been investigated in the past by studying natural cells and biomimetic cell models adhering on solid substrates. The amount of water in the membrane substrate gap, however, is difficult to determine. Here, we present a neutron reflectivity (NR) structural study of confluent epithelial cell monolayers on silicon substrates. In order to assure valid in-vitro conditions, we developed a cell culture sample chamber allowing to grow and cultivate cells under proper cell culture conditions while performing in-vitro neutron reflectivity measurements. The cell chamber also enabled perfusion with cell medium and hence allowed for contrast variation in-situ by sterile exchange of buffer with different H2O-to-D2O ratio. Contrast variation reduces the ambiguity of data modelling for determining the thickness and degree of hydration of the interfacial cleft between the adherent cells and the substrate. Our data suggest a three-layer interfacial organization. The first layer bound to the silicon surface interface is in agreement with a very dense protein film with a thickness of 9±2 nm, followed by a highly hydrated 24±4 nm thick layer, and a several ten nm thick layer attributed to the composite membrane. Hence, the results provide clear evidence of a highly hydrated intermediate region between the composite cell membrane and the substrate, reminiscent of the basal lamina.


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Introduction Epithelial cells adhere on the basal lamina, a thin, planar layer composed of extracellular matrix proteins, which supports all epithelia, muscle cells, and nerve cells. The basal lamina is a homogenous, macromolecular network formed mainly by collagens of type IV. The specific adhesion of the epithelial cells on the basal lamina is controlled by lock-and-key forces. Cell adhesion molecules such as integrins, which are exposed by the cell plasma membrane, form specific links to ligands exposed by the basal membrane. On solid substrates, epithelial cells form their own basal lamina-like structure by protein deposition. Cell adhesion on such surfaces is modulated by a competition between short range lock-and-key attraction forces due to specific binding, the long-range repulsions mediated by glycoproteins of the glycocalyx, and nonspecific forces due to the inorganic substrate. In this context, the cell substrate separation distance is an enduring matter of debate. For decades, the layer structure of cell adhesion has been studied, using model systems from supported lipid membranes to living cells. It has been shown that adhesion can be understood as wetting transition1 regulated by membrane elasticity1,2 as well as a variety of short- and longrange nonspecific forces1,3,4. These nonspecific forces include attractive van der Waals and electrostatic interactions, repulsive undulation forces due to thermally excited flickering of the lipid protein bilayer and a manifold of polymer induced forces1. The polymers can apply strong repulsive forces between the adhering interfaces. Short range lock-and-key forces mediated by cell surface receptors are responsible for the specificity of the adhesion process1,5,6. The wetting process results in the formation of adhesion domains, which allow cell adhesion at very low overall receptor densities. Studies on thermally excited bending undulations of lipid vesicles show that the membrane switches between weak and strong adhesion states, suggesting that adhesion is determined by a double-well interfacial potential with minima at a short and a long distance (in the case of the lipid vesicle hshort is approx. 10 nm and hlong is approx 40 nm)7–10. For immobile receptors, far fewer adhesion domains are observed 11. It is therefore assumed that adhesion domains formation is largely suppressed for immobile receptors, i.e. mobility of the receptors in the basal membrane is crucial and cannot be compensated by rearrangements in the plasma membrane. A schematic of a cell adhering to a surface by specific binding via lock-andkey mechanism in an adhesion domain and a region dominated by polymer repulsion is shown in Figure 1b, label (I) and (II), respectively.


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The majority of the structural studies on membranes on solid surfaces are based on optical microscopy techniques12–22, localizing the average cleft thickness at around 10 to 100 nm. Optical methods like RICM (reflection interference contrast microscopy) and FLIC (fluorescence interference contrast microscopy) encounter the problem that the real part of the refractive indexes of water and molecules hardly differs; thus no insight in hydration and hydration dynamics is provided. Since the wavelength of light is anyhow large compared to the dimensions of lipid membranes and proteins, which are in nano- or subnanometer range, it is difficult to resolve the ultrastructure of the interface region by optical interferometry. X-ray reflectometry, on the other hand, is well suited to analyze the structure of lipid bilayers at interfaces. However, also for X-rays, the scattering length of water, polymers, and proteins do not differ significantly. Furthermore, photochemical processes induced by X-rays can cause photo damage and alter cell shape and behavior. Neutron reflectometry (NR) is an alternative method for structural analysis of layered, hydrated soft matter systems23–25 with nm to even Å resolution. Neutrons scatter from the nucleus of an atom, therefore neutron scattering length varies for different isotopes. This can be exploited to determine the hydration of a sample by varying the H2O to D2O ratio. Since neutrons interact only weakly with matter, neutron reflectivity experiments can be done in transmission geometry, i.e. the neutron beam passes through the Si support prior to reflect from the solid/liquid interface, cf. Fig. 1a. Neutron reflectometry has been used intensively to study model membranes. Among others, the structure and hydrophobic region of supported lipid bilayers26–31, more complex and realistic model membranes like tethered supported lipid bilayers (SLBs)32, as well as lipid bilayers doped with peptides33 and natural lipid membranes extracted from bacteria34,35 were resolved using neutron reflectometry. In very few pioneering studies it has been shown that NR experiments on living cells are possible36,37, where the authors observed changes with temperature and shear force in single contrast experiments. However, the full potential of contrast variation in order to determine the profile and water gap of living cells has not been explored yet. Here, we investigate the cleft of confluent endothelial cell monolayer adhering to a SiO2 surface by neutron reflectometry. We exploit contrast variation between the cell membrane and the surrounding medium allowing for a detailed analysis of the thickness, the scattering length density profile, and in particular the hydration of the cleft between epithelial cell membranes and the substrate. For this purpose, we developed a cell culture sample chamber with controlled temperature, humidity and CO2 level suited for in-vitro reflectometry studies. The chamber allows for perfusion and exchange of buffer in order to vary the D2O/H2O ratio in the cell


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medium, see Figure 1c. The reflectometry data were analyzed by a minimal model in agreement with the framework of cell adhesion6. From the scattering length density of the various buffer media, we determined the hydration and the water exchange of the interface layers. Contrast variation strongly reduces the ambiguity of the data analysis. The results provide an estimate of the average cell density profile from a large ensemble of living cells across an interface and allowing us quantify the thickness of the hydration layer between cell membrane and substrate. Experimental results We present neutron reflectometry measurements on confluent cell monolayers cultivated on a solid Si substrate with a layer of native SiO2. The neutron reflectometry experiments require a specific sample environment, i.e. a dedicated sample chamber allowing for cell growth conditions, since eukaryotic cells demand controlled environment concerning sterility, temperature, and nutrition. Therefore, we developed and tested a sample chamber and established a measurement protocol suited for in vivo neutron experiments in a typical reactor hall like laboratory. A schematic of the sample chamber is depicted in Figure 1c. The sample chamber ensures sterility and temperature stability. The chamber is designed as a flow-throughsystem, allowing for a gentle exchange of the medium, avoiding shear forces and hence ensuring the necessary medium exchange for cell culturing and contrast variation. To exchange the medium, the chamber (reservoir dimensions: 80mm along the beam, 40 mm perpendicular to the beam, 5mm in height) was flushed with 120 ml, about 7 times the volume of the reservoir, within approximately 120 seconds. Exchange was done prior to the measurements, i.e. there was no flow during the data acquisition. Sterile packed tubing, connectors, and syringes were used in order to exclude contamination during liquid exchange for contrast variation. The syringes were pre-filled with cell medium and glued to the connectors under sterile conditions. Since the sample chamber is a sealed, CO2 free system, the cell medium was chosen accordingly. The temperature was kept constant at body temperature (37°C) at all times, i.e. from seeding to the final reflectometry experiments. On the instrument, a heated water bath (Julabo F12-ED) was connected to the sample chamber. During the transport from the cell culture laboratory to the instrument, the sample chamber was placed in an isolating box and kept warm using heating elements. A glass window provides optical access for control of the state of the cells. The total area accessible for the cells to adhere is about 30 cm2. Pictures of a cell layer growing to full confluence in the sample chamber used for the presented experiments can be seen in Figure 1d. Prior to seeding the cells, standard cell


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culture procedures were followed, please confer to Materials and Methods for more details on cell preparation and substrate cleaning. Neutron reflectivity data were acquired by recording the intensity of reflected neutrons R(q) versus the momentum transfer

q = 4 π (sin θ) / λ; here θ is the incidence angle and λ the

wavelength of the neutrons. The neutron reflectivity data presented here were acquired with the horizontal reflectometer38 REFSANS operated by HZG at MLZ and with the vertical reflectometer39 MARIA operated by JCNS at MLZ. For REFSANS, the wavelength spectrum λ ranged continuously from 2 to 10 Å, and four different angles were used (θ = 0.3°, 0.6°, 1.2° and 2.4°) for the time-of-flight mode. Measurements on MARIA were performed using two different wavelengths, 12 and 6 Å, respectively, with varying angle θ, i.e. in monochromatic mode. Resolution effects are included in the data analysis by using q/q= 0.05 and 0.1 for REFSANS and MARIA, respectively. Slit settings were chosen to ensure that the sample surface was not over-illuminated. Features in reflectivity data become much more distinct if the data are multiplied with q4, therefore some data are presented this way in the following to emphasis fine details in the modelling. In order to vary the contrast of the cell medium, medium was prepared in various D2O/H2O ratios of [90:10, 75:25, 50:50, 30:70, 15:85 and 0:100]. Pure D2O was omitted since the cells are not adapted to heavy water which acts systemic and at such high concentrations toxic. On REFSANS, cells were recorded in D2O/H2O ratios of [90:10, 75:25, 50:50 and 0:100] and on MARIA in D2O/H2O ratios of [75:25, 50:50, 30:70, 15:85 and 0:100]. Reference measurements of cell medium only (w/o cells) were recorded in [75:25] on REFSANS and in [75:25, 50:50, 30:70, 15:85 and 0:100] on MARIA. The neutron reflectivity of the cells recorded with REFSANS (data shown in gray color) are shown in Figure 2a. From these experiments, a region of interest was identified from 0 Å-1 up to around 0.04 Å-1. The experiments with MARIA focus on this region. Since a smaller q range allows for shorter measurement times, and thus for more contrast ratios, five contrasts could be recorded with MARIA which are shown in Figure 2b (gray symbols). The intensities are shifted vertically for clarity. The critical angle can be observed only when SLDMedium > SLDSi, i.e., for medium with a D2O content of 50% or higher, as expected for total reflection at the Si/water interface, confirming successful media exchange. Additional reference measurements (w/o cells) were also recorded for all contrasts. The signalto-noise ratio for MARIA is notably better, in part due to relaxed resolution compared to REFSANS; the resolution at REFSANS and MARIA was Δλ/λ = 0.05 and 0.1, respectively.


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Supporting figure S1a - f shows the reflected intensities of the cell layer of the experiments on MARIA (in color) compared to the reference measurement (dark grey) for each measured contrast. For most contrasts, distinct differences between the reference data and the cell data can be observed. This shows that the changes on the solid/liquid interface are induced by the presence of adhering cells and not due to cell medium only, confirming the sensitivity of the neutron reflectometry data towards presence of cellular interfaces. The largest intensity differences with and w/o cells are observed for a D2O content below or equal to 50%. Therefore, we focused on these D2O/H2O ratios in the MARIA experiment, which was second. For these conditions, reflected intensities are weak because the contrast between the substrate and the medium is strongly reduced, and total reflection vanishes. The inherent weak signal limits the accessible q-range. Data Analysis and modelling In the following, a self-consistent structure model is developed to analyze the neutron reflectometry data obtained for the different contrasts. Reflectivity scattering data cannot be inverted directly into SLD profiles due to missing phase information40. In order to extract the SLD profile for different contrasts, which contain the hydration profile as well, we employ a standard data modelling approach assuming a layered structure at the interface region between adhering cell and substrate. The data were analyzed using MOTOFIT41. The program allows for n discrete layers with individual SLD and thickness each to describe the stratified media. At the interface of two adjacent boxes, an error function takes roughness into account. The program converts the modeled SLD profile to a theoretical reflectivity curve using Abeles formalism42. This theoretical reflectivity curve is then compared to the reflected intensities and the SLD profile is varied until data and simulation match each other. This way, modelling yields thickness, roughness and SLD value of each layer. In order to avoid over-parameterization, a minimal model, i.e. as few as possible layers needed to reproduce the whole data set of different contrasts, is preferred. We do not expect the rough and fluctuating back-side membrane of the cells to influence the reflectivity experiment since there is no phase reference with the substrate because of limited coherence. We started by analyzing the reflectivity curves of the cells in two different contrasts [75:25 and 50:50] recorded on MARIA. The minimal model reproducing all reflectivity features consists of three finite thickness layers plus the semi-infinite substrate and bulk medium. One or two layer models did not yield a self-consistent fit of the multi-contrast data. Since reference measurements from bare wafers show no interference, we reason that the three layers are of biological nature. This model was iteratively applied to all remaining


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MARIA reflectivity curves in order to establish a self-consistent structure model in accordance with the data by variation of the SLD values while keeping layer thicknesses fixed. Finally, the three-layer model also was applied to the REFSANS data. Below, the differences in SLD values will be used to extract calculate hydration and density of the layers. The global fits are shown as solid lines in Figure 2 b. Given the large number of different contrast conditions, they are all in good agreement with the experimental data. To highlight the features of the specular reflectivity, the reflected intensities are data are multiplied with q4. The plots in Figure 3 a - e and f – g show the data recorded on MARIA and REFSANS respectively and the fits in more detail. The respective color codes are the same as in Figure 2. All modelling parameters can be found in the supporting information in Tables S1 (MARIA) and S2 (REFSANS). Also at larger q values, the model is in good agreement with the data [see data sets of 90:10, 75:25 and 50:50]. The SLD profiles corresponding to the modelled reflectivity curves for the MARIA and REFSANS data are shown in Figure 4. Adjacent to the substrate, we find a first layer, which is characterized by a small difference in SLD (ΔSLDLayer I) upon contrast variation, indicating little hydration and negligible proton exchange. However, the SLD of the next layer changes markedly after contrast variation, indication a high amount of hydration water accessible for efficient exchange. Finally, a third layer with intermediate hydration completes the sequence of layers, and with increasing distance from the interface the SLD values approach the bulk value. For the [50:50] REFSANS data, the first layer has the same SLD as the substrate due to contrast matching. The reduced bulk scattering length density value for deuterated mixtures may be due to protiated macromolecules within the cell with cannot be exchanged by contrast variation. Now we set the three layers obtained from our minimal, self-consistent model in a biological context. For this purpose, we assume the following: (1) the composite membrane of the adhering cell is a fluctuating protein-rich region; (2) complete exchange of medium SLDMedium, upon contrast variation, before starting a measurement; (3) for simplification, all biomolecular hydrocarbon layers are treated effectively as proteins, i.e. their scattering length density SLDProtein is supposed to change due to proton-deuterium (H/D) exchange as a function of SLDMedium as proposed by Efimova at al.43. For the calculation of the hydration and protein volume density from the SLD profiles, the following values for SLDProtein were used for layer II and layer III: SLDProtein = 2.9 * 10-6 Å−2 in cell medium dissolved in a D2O / H2O ratio of [90:10], 2.8 * 10-6 Å−2 [75:25], 2.3 * 10-6 Å−2 [50:50], 2.2 * 10-6 Å−2 [30:70], 2.1 * 10-6 Å−2 [15:85], and 2.0 * 10-6 Å−2 [0:100]. Layer I is little hydrated, and the labile hydrogens of the


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proteins do not exchange at higher D2O concentration. As the cells were grown in H2O, the SLDProtein is assumed to be 2.0 * 10-6 Å−2 in layer I for all contrasts. SLD profiles analyzed with these presumptions are shown in Figure 5a and b for MARIA and REFSANS respectively. This way, hydration (water fraction per volume) h and protein density per volumes can be calculated from the SLD profiles by a simple rule of proportion h = (SLDfit –SLDProtein) /(SLDcell medium – SLDProtein)44, cf. Fig. 5. To calculate the effective thickness, roughness, hydration, and protein volume density, as well as estimates for the variance, we average each layer over the values for all contrasts for each data set. The resulting average cell substrate density profiles are shown in Figure 5 as light blue and green area, respectively. The detailed values for thickness, roughness, and SLD for the three layers, as well as averaged values and error estimates of both data sets are detailed in supporting information T1. The resolution of a single reflectometry experiment can be estimated from the maximal q-value (q~0.1 Å-1), yielding here a resolution of 2/q ~ 6 nm. We perform independent reflectometry measurements at four to five different contrast for each sample. The thickness variance within one data set, i.e. for the same sample at different contrasts, is below the resolution limit for layer I and II. This is because we preselect for solutions with similar thickness values for all contrasts, in order to reject unphysical profiles. This implies that individual layer thicknesses should not vary with the D2O/H2O ratio. The interpretation of the resulting layer structure is shown in Figure 6. Layer I can be rationalized as a dense protein layer of 9±2 nm thickness with little hydration, which is located directly on the substrate. Comparison with the bare wafer in growth medium suggests that the cells form this layer actively. The second layer (II) exhibits a thickness of 24±4 nm, and a hydration value of 85±7%. The large hydration value indicates that water exchange is very effective in layer II upon contrast variation. The hydration and thickness values for layer II suggest that the cells are predominantly in the state of weak adhesion. Finally, layer III, which has an average thickness of 54±18 nm, and a huge variance, well beyond resolution effects, is associated with the composite membrane. Apparently, the composite membrane is much more difficult to quantify and subject to structural changes and deformation during different states of confluence, i.e. from sample-to-sample, while the first two layers (I and II) are rather well defined. This will be further discussed below in a biological context. Note that the SLD profile does not cover the whole cell, which extends several micron. The reason is twofold; first of all, the coherence length of the neutron experiment is limited well below a micron, therefore the upper side of the cell cannot interfere with the bottom. Furthermore, we expect the upper side to be even less aligned with a plane, therefore it is does


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not evoke its own reflection signal. In the analysis, we therefore infer the bulk scattering length density from fitting. The values obtained this way are close to, but not in full agreement with the nominal bulk values expected from H2O/D2O mixing (cf. supporting table T1). This deviation is not completely unexpected, since little is known about the neutron SLD inside cells upon contrast variation, i.e. to which extend organelles and macromolecules influence the SLD. Discussion We measured the scattering length density profile across the interface of living cells adjacent to a Si substrate by neutron reflectometry contrast variation. Modelling, using a minimal model for discrete layers, reveals best agreement with a three-layered structure. The contrast variation allows for resolving the thickness and hydration of these layers and confirms efficient water exchange in the second layer. We suggest that this second layer is a central element of the so-called cleft, providing ion conductivity inherent to cell function. One of our main findings is that this cleft is about 20 nm thick and 80 to 90% hydrated for the experimental conditions presented here. We also comment on the signature of the composite membrane. The standard model of cell adhesion assumes that the cell membrane flickers due to thermal excitation. Bruinsma et al. have shown in a study with biomimetic cell models that weakly adhering regions of the membrane have a fluctuation amplitude of about 20 nm. For cells, it was initially assumed that due to the strong coupling of the plasma membrane to the actin cortex, thermally induced undulation forces are not significant. However, Zidovska et al. have shown in studies with macrophages that nucleated cell envelopes exhibit pronounced bending excitations of around 10 nm root mean square amplitude. Further, in a living cell, approximately 50% of the plasma membrane mass is protein45. This suggests that the composite membrane that adheres to the surface, including the 4 nm thin lipid bilayer, is smeared out vertically as confirmed by our analysis. Previous pioneering experiments on epithelial cells under dynamic flow conditions, but without contrast variation, were in agreement with a rather well defined lipid membrane signature. Our model differs in this respect as it does not presume a distinct lipid membrane entity but rather a composite membrane with a smeared scattering length density profile. Finally, it needs to be emphasized that the presented neutron reflectometry experiments average over an area of around 30 cm2, i.e., about 107 cells. Thus, the measurement averages the dynamics and the structure of an extremely large number of cells while experiments with optical microscopy mostly are done on few individual cells. However, endothelial cells behave


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differently when isolated from their confluent, native environment, which is a strong argument for the kind of measurements with confluent layers presented here. In this paper, it is shown that already the most simplistic structure model benefits from the sensitivity of neutrons for different hydrogen isotopes for analyzing the thicknesses and the hydration profile of the interface between the cell and the substrate. New developments of sophisticated microscopy and X-ray scattering techniques22,46,47 are highly promising to provide detailed structural insights in cell adhesion processes. Nevertheless, neutron reflectometry contributes to answering questions on subtle structural changes at the cell substrate interface, especially regarding hydration. Furthermore, deuteration of cellular components involved in adhesion, e.g. hyaluronic acid or lipids, by the use of proper culture conditions, may allow for a much more detailed analysis of the molecular distributions and substructures involved48. MATERIALS AND METHODS Neutron reflectometry:

Cell line: A cell layer that does not change its structure significantly over time when reaching confluence or even over-confluence is a requirement for recording NR data as cell growth naturally will continue in the sample chamber. Measurements were performed on a confluent cell layer as the intensity of the reflected signal is lower for lower cell surface densities36. MDCK (Madin-Darby Canine Kidney) cells were chosen for the presented NR experiments. Their strong adhesion allows for transporting the adherent cells to the reactor guide hall without detaching. Furthermore, the MDCK cell layer does not detach when reaching confluence. MDCK cells continue cell division, and a reduction of cell size can be observed. Mitotic arrest is achieved once the cell area reaches a certain threshold49. Cell culture: MDCK-II cell line was cultured in minimal essential medium (c-c-pro) containing 2mM Lglutamine and 10% fetal calf serum at 37°C. After growing to around 80% confluence, they were trypsinated and centrifuged at 1000 rpm for 3min. Resuspension of the cell pellet was done with cell medium. For the experiments, cells were resuspended in Leibovitz’s L15 medium powder with 2mM L-glutamine and 10% fetal calf serum solved in either only H2O or a mixture of D2O and H2O. In the neutron sample chamber cells were seeded with around 50% cell density. One day after seeding cells, and after reaching full confluence, medium was ACS Paragon Plus Environment

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exchanged in cell culture laboratory under sterile conditions. Note that cells were initially cultivated in H2O as buffer. Cells remain viable during the course of the experiments, which takes typically two days for 4-5 contrasts. Sample preparation: Silicon blocks were cleaned in ultrasonic bath in chloroform, acetone, ethanol and purified water for 10 minutes each step. To sterilize all components of the chamber, they were stored in 75% ethanol solution for 1h under sterile conditions. Afterwards, they were flushed with purified water and dried under sterile conditions. Prior to seeding of the cells, the mounted chamber was filled with medium and incubated for ~30 min.

ASSOCIATED CONTENT Supporting information The Supporting Information is available free of charge on the ACS Publications website. Neutron reflectometry data of the cell layer vs. reference measurements of only cell medium, Modelling parameter for experiments on MARIA and REFSANS AUTHOR INFORMATION Corresponding Author B.N. Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This work has been funded by the BMBF (Project 05K13WM1) and by the Deutsche Forschungsgemeinschaft via SFB1032 (Project A7). We thank Ida Berts for support during the REFSANS beamtime, Gerlinde Schwake for valuable advice on cell culture, Ann Junghans and Luka Pocivavsek for fruitful discussions about data analysis, and Adrian Rennie and Andrew Nelson for helpful communication about data modelling. Furthermore, we thank the MLZ management and staff at MARIA and REFSANS for continuous support.


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Figure 1 (a) The scattering geometry; a neutron beam (black dashed arrow) is reflected from a cellular layer (green) at the solid-liquid interface of a Si block (grey). (b) Schematic close up of an epithelial cell (green) adherent to the basal laminar (grey); specific adhesion is mediated via lock-and-key interaction (I), polymers exert repellent forces (II). (c) Design principle of the sample chamber; the PTFE piece and the silicon wafer are clamped together by an aluminum frame. A channel allows for cycling of heated water from a water bath. An O-ring inserted in the PTFE ensures water tightness. In- and outlets allow for exchanging the cell ACS Paragon Plus Environment

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medium in the reservoir. Optical access is granted by a transparent window that is glued to the PTFE. (d) Optical micrograph of a layer of MDCK cells growing to confluence in the sample chamber used for experiments on REFSANS and MARIA.

Figure 2 Neutron reflectometry data. Grey symbols show normalized reflected intensities vs. momentum transfer q. Modeled reflectivity curves are shown as solid curves. The curves are shifted vertically for clarity. (a) REFSANS reflectometry data of the same cell layer recorded in cell medium dissolved in a D2O/H2O ratio of [90:10] (squares, purple), [75:25] (circles, black), [50:50] (empty squares, blue) and [0:100] (empty circles, orange). (b) MARIA reflectometry data of the same cell layer recorded in ratios of [75:25] (squares, black), [50:50] (circles, blue), [30:70] (diamonds, red), [15:85] (empty circles, green) and [0:100] (empty squares, orange).


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Figure 3 Reflectometry data (grey symbols) and modelled reflectivity curves (solid lines) for cell medium dissolved in a D2O/H2O ratio of (a) [75:25] (black), (b) [50:50] (blue), (c) [30:70] (red), (d) [15:85] (green) and (e) [0:100] (orange) measured on MARIA and (f) [90:10] (purple), (g) [75:25] (black), (h) [50:50] (blue) and (i) [0:100]CM0 (orange) measured on REFSANS.


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Figure 4 Scattering length density profiles used to calculate the simulated intensities in Figure 2 and 3. Color code is the same. Dotted grey lines represent lower and upper values of the variation of the bulk SLD due to the D2O/H2O ratio. The SLD range of a protein layer (SLDProtein), which depends on the D2O/H2O ratio only via hydrogen exchange, is indicated by the striped grey area.


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Figure 5 Hydration and protein volume density volume profiles calculated from the SLD profiles from the data recorded on (a) MARIA and (b) REFSANS. The overall hydration and protein volume density curves, shown in light blue and green respectively, are the average of the profiles calculated for each contrast and depicted in color.


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Figure 6 Interpretation of the NR experiments. The substrate is coated with a dense protein layer (layer I), followed by a highly hydrated layer II. Layer III represents the composite membrane. At large distance from the substrate, the hydration transitions to 100%. Average values for layer thickness and hydration resulting from the analysis of the data recorded on MARIA and REFSANS can be found in the text.


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Thickness

Roughness

(nm)

(nm)

Layer I

11±2

3

16

Layer II

27±1

10

90

Layer III

70±11

16

53

Thickness

Roughness

Hydration

(nm)

(nm)

(%)

Layer I

7±1

1

12

Layer II

21±3

10

80

Layer III

41±6

14

31

MARIA

REFSANS

Hydration (%)

Tabular 1 Average values for thickness, roughness, and SLD for Layer I - III for MARIA and REFSANS data. Error bars report the variance from averaging thickness from different contrasts (cf. SI, Table 1). The overall average thickness value for layer I and II is 9±2 nm and 24±4 nm, respectively. Errors indicate variance from averaging different samples.


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ToC-Figure:


Probing the Interface Structure of Adhering Cells by Contrast Variation

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