Article pubs.acs.org/Langmuir
Induction of Intermembrane Adhesion by Incorporation of Synthetic Adhesive Molecules into Cell Membranes Ai Ushiyama,†,‡ Mio Ono,†,‡ Chiho Kataoka-Hamai,† Tetsushi Taguchi,† and Yoshihisa Kaizuka*,† †
National Institute for Materials Science, International Center for Materials Nanoarchitectonics, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047, Japan ‡ School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan S Supporting Information *
ABSTRACT: Modulation of cell adhesion by synthetic materials is useful for a wide range of biomedical applications. Here, we characterized cell adhesion mediated by a semisynthetic molecule, cholesteryl-modified gelatin (chol-gelatin). We found that this hybrid molecule facilitated cell adhesion by connecting two apposed membranes via multiple cholesterol moieties on the gelatin molecules, whereas unmodified gelatin did not bind to cell membranes. Analyses revealed that the rate of the formation of cell adhesions was increased by displaying more cholesterol moieties on the cell membrane. In contrast, the area of the cell adhesion site was unchanged by increasing the number of cholesterol molecules, suggesting that chol-gelatin may suppress cell spreading. Such restriction was not observed in cell adhesion mediated by the mutant of physiological adhesion protein CD2, which lacked its cytoplasmic domain and was unable to connect to cytoplasmic actin filaments, but had a similar affinity for its ligand compared with the chol-gelatin−cell membrane interaction. Further analysis suggested the restriction of cell spreading by chol-gelatin was largely independent of the modulation of the surface force, and thus we hypothesize that the restriction could be in part due to the modulation of cell membrane mechanics by membrane-incorporated chol-gelatin. Our study dissected the two roles of the hybrid molecule in cell adhesion, namely the formation of a molecular connection and the restriction of spreading, and may be useful for designing other novel synthetic agents to modulate various types of cell adhesions.
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surface force.16 In contrast, the adhesion of actual cells is regulated by specific binding of proteins. Although the binding of most of these adhesive molecules is reversible, the kinetics of the association and dissociation are often asymmetric, resulting in binding with a low dissociation constant, which is spatially sparse and dynamic owing to diffusion. Thus, the physiological process deviates from the simplest wetting model, and characterization of individual adhesion proteins is critical.15 Another level of complexity is that cell adhesion molecules are linked to cellular biochemistry including the actin cytoskeletal network. Binding of adhesion molecules such as integrins induces actin polymerization, while polymerization of the actin network modulates cellular deformation by protrusion or lamella formation, which alters the adhesion site area. Moreover, cortical actin filaments regulate cell membrane mechanics. Cell adhesion must be understood through all of these interacting factors at both molecular and cellular levels. On the basis of the physical chemistry of cell adhesion, we characterized synthetic cell adhesion molecules that may have useful applications. Our model molecule is a semisynthetic hybrid molecule, cholesteryl-modified gelatin (chol-gelatin). Gelatin is derived from thermally denatured collagen molecules, which is an abundant natural resource with biocompatibility.
INTRODUCTION Cell adhesion regulates a wide spectrum of biological processes. Cells express numerous types of adhesive molecules to facilitate specific cell−cell contacts in immune and neuronal systems,1,2 create specialized structures such as tight and gap junctions, and regulate the dynamic cellular locomotion in the immune system and developmental processes.3,4 Cancer metastasis may also involve changes in the modes of cell−cell adhesion.5 Modulation of these physiological adhesion processes by synthetic materials is a promising approach for future therapeutics, but it is still a challenge with current technologies.6−8 For example, a simple strategy using bulk biomaterials such as hydrogels to force cells into a connective state has significant problems such as defects in nutrient supply and subsequent cell death in the core of the cell mass.9 Physiological adhesion processes are regulated by dynamic and specific interactions of multiple adhesion proteins as well as other factors such as cellular mechanics.10−12 Thus, the modulation of such complicated processes by synthetic materials may require a better understanding of how these multiple factors are regulated during adhesion processes. Such complex regulation of cell adhesion has been characterized from a physical chemistry perspective.12−17 In simplest terms, cell adhesion is described as a wetting process regulated by a balance between surface attraction/repulsion and the elasticity of cells.18 This model explains the adhesion of synthetic lipid vesicles that have a uniform and continual © 2015 American Chemical Society
Received: November 20, 2014 Revised: January 22, 2015 Published: January 23, 2015 1988
DOI: 10.1021/la504523c Langmuir 2015, 31, 1988−1998
Article
Langmuir Thus, it is ideal as a backbone biomaterial.19−21 In addition, gelatin tolerates organic solvents and can undergo extensive chemical modification.19−21 Conjugation of cholesterol has been applied for recruitment of various molecules to membranous structures.22−24 Using a single-cell level assay, we demonstrated that this semisynthetic molecule facilitated membrane−membrane binding. The rate of binding was enhanced by increasing the total number of cholesterol moieties displayed on the cell surface, suggesting that the binding of cholesterol to lipid bilayers facilitates membrane− membrane binding. In contrast, the cell adhesion site was limited to a small area, and cells did not spread with an increased number of cholesterol moieties for binding. These results reflected the two distinct properties of the hybrid cholgelatin molecule: strong affinity to lipid membranes mediated by cholesterol moieties and a repulsive effect created by the whole molecule. Our study dissects these two roles of cholgelatin in the process of cell adhesion and may help in the design of other hybrid molecules with various combinations of molecules to control cell adhesion.
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bovine serum (FBS), and S2 cells were maintained in Schneider’s Drosophila Medium (Life Technoliogies). The chimeric fusion of CD2EM and mCherry genes was cloned, and JCaM2 cells were transfected by electroporation.26 CD58 were purified from red blood cells, labeled with Alexa 647 succinimidyl ester (Life Technologies), and incorporated into liposomes as described previously.26 Microscopy and Image Analysis. Cells and supported bilayers were imaged by the Leica (Solms, Germany) AF6000LX total internal reflectance (TIRF) microscope equipped with a 100× 1.46 NA oilimmersion objective and a Cascade II EMCCD camera (Roper, Tuscon, AZ). For RICM imaging, a filter cube consisting of a narrowband-path filter (543-553 nm) and a beam splitter was used with arclamp illumination. Densities of chol-gelatin proteins were calculated through multiple steps. First, cells preincubated with 1.0 mg/mL chol-gelatin doped with 4% of Oyster 647 labeled chol-gelatin were plated on PLL surface, and the images of the cells were taken in the near-infrared filter (Ex 620/60, Em 700/75 nm). These images were calibrated with fluorescence standards, as described previously.27 Standards were created from the images of bilayers containing fluorescent molecules (0.02−0.25% DiD) recorded through the same near-infrared filter. To directly compare protein images with membrane standards, protein (Oyster 647 labeled chol-gelatin) and membrane dye (DiD) fluorescence were compared by fluorometry (F-7000 fluorescence spectrophotometer, Hitachi, Tokyo, Japan). Protein and dye emission spectra in buffer were obtained at excitation and emission wavelengths of the microscopy filter to establish a baseline. Integrated fluorescence intensities were calculated from 2D spectra and the arc lamp spectrum of a microscope. The ratio of the integrated intensities between the protein and the membrane dye was used as the scaling factor in the calibration.27 After establishing the calibration for microscopy images, the calculated density data were compared with the signal obtained by flow cytometry. The average chol-gelatin density for cells preincubated with 1.0 mg/mL chol-gelatin, obtained by the microscopy-based calibration, was then compared with the fluorescence intensity signals for those cells obtained by flow cytometry. Using this ratio of these microscopy and flow cytometry signals, together with the signal of cells in the absence of chol-gelatin incorporation, all other flow cytometry signals were converted to the density of chol-gelatin, by assuming the fluorescence signals obtained by flow cytometry was linear to the amount of dyes. Area and diameter of adhesion were calculated from RICM images through semiautomatic data processing, though background subtraction and thresholding using ImageJ (National Institutes of Health, Bethesda, MD). Flow Cytometry. An SP6800 spectral analyzer (SONY, Tokyo, Japan) was used for flow cytometry analysis. Jurkat cells in HBS were incubated with different concentrations of (chol)n-gelatin (n = 0, 0.50, and 8.75) doped with 4% of Alexa488-labeled (chol)n-gelatin (n = 0, 0.50, and 8.75) at 25 °C. After the cells were incubated with cholgelatin for designated time periods and then excess chol-gelatins were washed out with a brief spin, cells were resuspended in HBS and were injected to the analyzer.
MATERIALS AND METHODS
Materials. Gelatin from tilapia fish skin (G-2708P, average molecular weight ∼70 kDa, isoelectric point ∼5.5) was obtained from Nitta Gelatin (Osaka, Japan). Cholesteryl chloroformate was obtained from Sigma (St. Louis, MO). Oyster 647-tetrafluorophenyl ester (TFP) and Alexa 488-TFP esters were obtained from Boca Scientific (Boca Raton, FL) and Life Technologies (Carlsbad, CA), respectively. 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) was from Avanti Polar Lipids (Alabaster, AL), and 1,1′-dioctadecyl3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) and 1,2dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine, triethylammonium salt (TR-DPPE), were purchased from Life Technologies. The chol-gelatin molecule was produced by modification of gelatin with cholesterol as described previously, through the reactions between primary amines in gelatin and cholesteryl chloroformate,25 and the degree of modification (the average cholesterol per molecule) was calculated through the quantification of free amines by the trinitrobenzenesulfonic acid method.25 Two different samples of chol-gelatin were prepared: the low cholesterol batch (modification of average 4% of primary amines in gelatin or average 0.5 cholesterols conjugation per gelatin molecule) and the high cholesterol batch (modification of average 70% of primary amines in gelatin or average 8.75 cholesterols conjugation per gelatin molecule). Cholesterol modified gelatin was further labeled with fluorescent dye conjugated TFP esters, and labeling efficiency (0.2−0.3 dyes per gelatin molecule) was determined by absorbance measurements using a spectrophotometer at the maximum absorption wavelengths for the fluorophores (together with the information describing the molecular extinction coefficients provided by the manufacturers) and at 230 nm for gelatin. Poly-L-lysine (PLL)-coated coverslips were obtained from Matsunami (Osaka, Japan). Jurkat cell, JCaM2 cell, and S2 cell were obtained from DS pharma (Tokyo, Japan), Dr. A. Weiss (UCSF), and Life Technologies, respectively. Cells and Supported Lipid Bilayers. Large unilamellar vesicles containing of 0.05% of TR-DHPE or 0.02−0.25% of DiD and DOPC were produced by extrusion. Cleaned coverslips etched by 5 M NaOH for 4 h were rinsed with water, and then vesicles were deposited on the etched glass surface under HBS (Hepes buffer saline). After washing with the HBS buffer containing 1 mM CaCl2 and 0.5 mM MgCl2, a single fluid supported lipid bilayer was prepared on the glass surface. Supported bilayers were used within 10 min after the prep to avoid formation of any defect that induced nonspecific cell adhesions. Cells were incubated in HBS containing chol-gelatin and then either directly deposited on the bilayers/PLL coated glasses or first washed out to remove free chol-gelatin before the deposition. Jurkat and JCaM2 cells were maintained in RPMI 1640 media supplemented with 10% fetal
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RESULTS AND DISCUSSION Incorporation of Chol-Gelatin into Cell Membranes. We evaluated tilapia fish, skin-derived gelatin molecules modified with cholesterols (chol-gelatin) as a model synthetic material to modulate cell adhesion. Chol-gelatin is potentially useful as a tissue adhesive via cross-linking and gelation.25,28 In this study, we characterized the soluble form of this semisynthetic chol-gelatin molecule and examined whether it can connect two apposed membranes via interactions between the cholesterol moieties and lipid bilayers. We did not observe gelation of the molecule under our experimental conditions, and chol-gelatin behaved as a soluble polypeptide. We used suspension cell lines to better differentiate the adhesion mediated by chol-gelatin from nonspecific cell adhesion. 1989
DOI: 10.1021/la504523c Langmuir 2015, 31, 1988−1998
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Figure 1. Chol-gelatin incorporation into cell membranes. (A−C) Bright field and epifluorescence microscopy images of Jurkat cells with incorporation of Alexa 488-labeled (chol)n-gelatin. n = 0 (A), 0.50 (4% primary amines, B), and 8.75 (70% primary amines, C). (D, E) Representative flow cytometry data of Alexa 488-labeled chol-gelatin incorporation into cell membranes. Fluorescence signals from cells incubated with (chol)8.75-gelatin for 1 h at various concentrations (4.0, 0.5, 0.1, and 0.025 mg/mL) (D) and cells with competitive binding at 4.0 mg/mL for 1 h (E) were compared with data from cells incubated with unmodified gelatin (gray in parts D and E). (F, G) Dynamics of chol-gelatin incorporation into cell membranes calculated from temporal flow cytometry analysis and fluorescence calibration. Temporal dynamics of (chol)n-gelatin incorporation [n = 8.75 (F) and 0.50 (G)] at various concentrations are plotted in left panels. Competitive binding of both (chol)n-gelatin samples was examined by replacing labeled chol-gelatin in solution with unlabeled molecules at the same concentration (right panels). (H) Saturation levels of chol-gelatin densities at 1 h of incubation in (F) and (G) for all conditions were plotted along with best fit exponential curves. (I) kobs obtained from exponential fits in (F) and (G) were plotted along with the various concentrations. Error bars represent the SEM.
To confirm whether the chol-gelatin molecule interacted with cells, we imaged Jurkat T cells incubated with fluorescently labeled chol-gelatin. Jurkat cells are a suspension cell line but can adhere to and spread on its target cells under biological stimulation.29,30 After incubation for 1 h with 4.0 mg/mL cholgelatin with various degrees of cholesterol modifications ([chol]n-gelatin, n = 0, 0.50, and 8.75) and washout of excess chol-gelatins, the gelatins with both low and high degrees of cholesterol modifications (n = 0.50 and 8.75) were localized in the cell membranes (Figure 1B,C). Endocytosis of chol-gelatin was not detectable at either 37 °C (Figure 1A−C) or 25 °C (data not shown), suggesting that chol-gelatin interacts with lipid bilayers but not cytoplasmic components including actin
cytoskeletons. In contrast, we did not detect incorporation of unmodified gelatin (n = 0) into cell membranes at 37 °C (Figure 1A) or 25 °C (Figure 1D−G). These results demonstrate that unmodified gelatin molecules do not have an affinity for cell membranes. However, as expected, small cholesterol moieties mediate rapid incorporation of the whole chol-gelatin molecule into cell membranes. Note that the heterogeneity in fluorescence signals among cells (Figure 1B,C) is within the distributions of fluorescence signals detected by flow cytometry (Figure 1D,E) but may be also exaggerated due to imaging conditions (e.g., background fluorescence from cells out of the focal planes and inhomogeneous illumination). The standard error of the mean (SEM) of the signals from each 1990
DOI: 10.1021/la504523c Langmuir 2015, 31, 1988−1998
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Figure 2. Chol-gelatin mediates cell-bilayer adhesion. (A) Schematic of the model cell adhesion system consisting of live cells and supported lipid bilayers. (B) Imaging of cell-supported lipid bilayer (SLB) adhesion mediated by chol-gelatin. Epifluorescence image of chol-gelatin (top left), bright field (top right), RICM (middle left), and epifluorescence image of Texas Red-DPPE in supported bilayers (middle right). Asterisk indicates a dead cell with high autofluorescence. (C) Enlarged images of the adhesion site and fluorescence intensity along the line on images. (D) Rates of cell-SLB adhesion formation of cells that were preincubated with various concentrations of (chol)n-gelatin molecules (n = 0, 0.50, and 8.75). Results were compared with cells without any (chol)n-gelatin. Cells incubated with 4.0 mg/mL (chol)n-gelatin (n = 0.50 and 8.75) were also compared in terms of adhesion on freshly prepared SLBs and SLBs preincubated with 4.0 mg/mL (chol)n-gelatin for 1 h (marked “SLB” and in pink and cyan lines). (E) Cell adhesion at 3 min in (A), except for cells with no chol-gelatin, (chol)n-gelatin (n = 0), and SLB with preincubated (chol)n-gelatin (n = 0.50 and 8.75; marked “gelatin-SLB”) at 5 min (A). (F) Ratios of the diameters of cell−SLB adhesion sites and diameters of cells with incorporation of (chol)n-gelatin (n = 0.50 and 8.75) at various concentrations. Error bars represent the SEM.
membranes by comparing fluorescence images of chol-gelatin in cell membranes adhered to plates and images of fluorophores of defined densities in supported lipid bilayers (see Materials and Methods). In addition, we found that the incorporation of both (chol)n-gelatins (n = 0.50 and 8.75) into cell membranes was saturated at a high concentration (∼4.0 mg/mL). Furthermore, regardless of the number of conjugated cholesterols per molecule, the saturated densities of (chol)ngelatins were similar (∼7000 molecules/μm2). We measured the dissociation constant (KD) for the interaction between (chol)n-gelatin and the Jurkat cell membrane by fitting exponential curves to the saturation binding curves (Figure 1H). The KD was 18.7 μM (chol0.5gelatin) and 6.86 μM (chol8.75-gelatin). Rate constants (kon and koff) for the interactions were calculated from linear fits to kobs (Figure 1I). kobs was calculated from the time constants of binding curves for the first-order binding kinetics approx-
sample in the cytometry data (∼1000 cells per sample) was in the range of 1/20−1/30 of the mean (data not shown). We used flow cytometry to measure the affinity of cholgelatin for cell membranes (Figure 1D−G). A rapid increase in the fluorescence of cells was detected over a wide range of cholgelatin concentrations (0.025−4.0 mg/mL), confirming a high association rate of chol-gelatin in the cell membrane (Figure 1D−G). However, we also observed rapid dissociation of cholgelatin from cell membranes after washing out excess cholgelatin molecules (data not shown). Thus, we assessed this dissociation through a cytometry-based competitive binding assay by replacing free chol-gelatin in solution with unlabeled chol-gelatin after saturation of binding at 1 h of incubation. As a result, we confirmed rapid and continuous exchanges of cholgelatin molecules between cell membranes and the surrounding medium (Figure 1F,G). The fluorescence signals were calibrated to the density of chol-gelatin molecules in cell 1991
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charged fluorescently labeled lipids were doped. In the absence of any specific binding molecules incorporated into the supported bilayers, Jurkat cells did not adhere to the lipid bilayers when the bilayers were fresh and without any degradation or surface defects (data not shown). Jurkat cells were incubated with 10 mg/mL (chol)8.75-gelatin for 1 h and then applied to the supported lipid bilayers in buffer together with free chol-gelatin at 1 mg/mL (∼14 μM). In this manner, the induction of cell−cell adhesion was at least in part mimicked. Soon after incubation, fluorescence microscopy revealed spots of labeled chol-gelatin at the interface of the cells and lipid bilayers (Figure 2B, upper left). To examine whether the cells with fluorescent chol-gelatin spots were adhered to the lipid bilayers, we imaged the cells by reflection interference contrast microscopy (RICM, Figure 2B, bottom left). RICM imaging maps the cell adhesion sites as dark spots that are created by interference of light reflected from cell membranes and surfaces.38 We detected the cell adhesion on bilayers by RICM, and the positions of these adhesion sites matched those of the fluorescent chol-gelatin spots (Figure 2B). In the absence of chol-gelatin, no cell adhesion was detected by RICM (data will be discussed in the next section). We also confirmed that the cell adhesion did not alter the macroscopic structures of the supported lipid bilayers by imaging fluorescent lipids doped in the bilayers (Figure 2B, bottom right), while microscale integrity in the bilayers may be perturbed. These data suggest that chol-gelatin induces cell adhesion to supported bilayers. The fluorescence intensity around the cell−bilayer interface of adhered cells was darker than the background signal outside of the cells, except for the bright fluorescent spots in the center corresponding to the adhesion site probed by RICM (Figure 2C). The high background signal was due to the fluorescence of chol-gelatin incorporated into the supported bilayers because incorporation of chol-gelatin into synthetic lipid bilayers and cell membranes was similar.39 We lowered the background fluorescence by washing out excess chol-gelatins, imaged the interface between cells, and supported bilayers by total internal reflection fluorescence (TIRF) microscopy (Figure S1A) and found that the fluorescent chol-gelatins incorporated into the bottom of cell membranes appeared at the interface region. On the basis of these results, we concluded that the fluorescent spots shown in Figure 2B (top left) were due to fluorescence from chol-gelatins incorporated into the membranes of adhered cells. In contrast, other nonadherent cells were floating near the surface by balancing with gravity. We speculated that chol-gelatin molecules bound to the supported bilayer below the nonadherent cells competed with chol-gelatin in cells for binding to the supported bilayers, which prevented adhesion. Nonetheless, many cells were adhered to the bilayers because, when the cells sank to the bilayer quickly, the bilayers below these adhered cells were protected from excessive incorporation of free chol-gelatin, and accordingly the fluorescent signal there was lower than the outside. The structure of the adhesion sites was not fixed permanently, probably because of rapid association and dissociation of chol-gelatins in the membranes. The dynamics of the adhesion sites were recorded by time-lapse RICM images, and submicron-scale steps of diffusive motion at every 2 s were plotted (Figure S1B). It also shows that any local structural constraints in lipid bilayers that could be induced by the insertion of cholesterol was not strong enough to interfere with such macroscopic dynamics of the cells. In contrast, floating cells without any adhesion site diffused at much longer
imation (Figure 1F,G). However, these curves were not well fitted to first-order reactions at higher concentrations of cholgelatin (Figure 1F,G). Accordingly, the kobs plot was not linear at higher chol-gelatin concentrations (Figure 1I). Nonetheless, koff should be in the range of 0.10−0.20 s−1 and kon should be 0.01−0.07 M−1 s−1 for both chol0.5-gelatin and chol8.75-gelatin (Figure 1I). These results demonstrate that the binding of cholgelatin to the cell membrane is weak compared with typical receptor-ligand binding. KD and koff were similar for both (chol)0.5-gelatin and (chol)8.75-gelatin, and the saturated level densities of both cholgelatins in cell membranes were similar. Thus, the binding of chol-gelatin to the cell membrane is most likely mediated by a single cholesterol moiety, even when a chol-gelatin molecule has multiple cholesterol moieties. Although the binding of (chol)8.75-gelatin was slightly better than that of (chol)0.5-gelatin in the intermediate range of gelatin concentrations (0.1−1.0 mg/mL), we speculated that this slight enhancement was not due to the difference in the binding affinities of (chol)0.5-gelatin and (chol)8.75-gelatin, but rather the difference in effective concentrations. Because a substantial amount of (chol)0.5gelatin molecules did not carry even a single cholesterol moiety, the concentration of chol-gelatin that had a cholesterol and could bind to the cell membrane was always lower for (chol)0.5gelatin than (chol)8.75-gelatin. Previous studies reported the ratios of the radius of gyration to hydrodynamic radius for various gelatin molecules to be 1.5−1.8,31−33 suggesting that the conformation of gelatin in solution is linear rather than globular, and only 2.5% of tilapia gelatin amino acid content is lysine whose primary amine was used for the cholesterol conjugation.34 Such conformation and structure of the (chol)8.75-gelatin may be different from those of many physiological membrane-anchored proteins (e.g., Src, Ras, or Rab family proteins or α subunits of trimeric G proteins (Gα)35). These proteins can stably interact with membranes via two or more lipid anchors that are located in close proximity in random coils at the N- or C-terminus. However, more structural information on membrane-anchored (chol)8.75-gelatin is required to understand how the protein−membrane anchors are formed and regulated. Membrane density of chol-gelatin in our experimental conditions was estimated to be 100−7000 molecules/μm2. This is equivalent to the density of 0.007%−0.5% of fully packed lipids in a liposomal surface, ∼1 400 000/μm2, and Jurkat cell membranes (inner and outer leaflets) contain an abundance of cholesterol.36 However, a substantial fraction of the cell membrane surface is occupied with proteins, and the inserted cholesterol of chol-gelatin was not trafficked internally to balance the plasma membrane contents. Thus, the insertion of cholesterol derived from chol-gelatin alone may still have an impact on the cell membrane structures that may be substantial enough to induce a condensing effect that was observed in liposomes.37 Intermembrane Binding Mediated by Chol-Gelatin. To investigate whether chol-gelatin mediates cell adhesion, we monitored cell adhesion to substrate-supported lipid bilayers as a model system for cell−cell adhesion (Figure 2A). Vesicle fusion to a silica surface created a single fluid lipid bilayer, and the lipid bilayer-cell interface mimicked the structure of cell− cell binding. In this system, we could quantify cell adhesion at the single cell level by microscopy. The supported bilayers consisting of phosphatidylcholines and the net surface charge of membranes were nearly neutral, while 0.05% of negatively 1992
DOI: 10.1021/la504523c Langmuir 2015, 31, 1988−1998
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the affinity of the molecule for cell membranes may resolve this issue and induce more cell−cell adhesion. Gelatin Incorporation into the Cell Membrane Suppresses Cell Spreading. To study the negative effect of chol-gelatin on cell spreading, separately from its positive effect mediated by the interactions between cholesterols and lipid bilayers, we analyzed the adhesion of cells incorporating cholgelatin on a nonmembranous surface, standard poly-L-lysine (PLL)-coated glass. In the absence of chol-gelatin, Jurkat cells adhered and spread on the PLL-coated surface (Figure 3A,B). However, cells preincubated with 4.0 mg/mL of both (chol)0.50gelatin and (chol)8.75-gelatin exhibited a significantly smaller adhesion area on the PLL-coated surface than the untreated cells (Figure 3A,B). Such suppression was dependent on the density of chol-gelatin on the cell membranes and was substantially alleviated when the concentration of chol-gelatin was decreased to 0.5 mg/mL (Figure 3B). These results demonstrate that the incorporation of chol-gelatin suppresses cell spreading, regardless of the nature of the adhesion, through either specific molecular binding on supported bilayers or nonspecific adhesion on PLL. We also analyzed the effect of cytoplasmic actin filaments. Two forms of cytoplasmic actin filaments are involved in the processes of cell adhesion and spreading. The first form is cortical actin filaments that modulate the two-dimensional viscosity of cell membranes and the dynamics of adhesion,11 and the second form is actively polymerizing filaments at the leading edge of lamella, which can induce cell deformation and further cell spreading.40,41 Jurkat cells have a strong actin polymerization activity upon biological stimulation, which induces spreading on their target cells.29,30 To depolymerize cytoplasmic actin filaments, we treated the Jurkat cells with latrunculin B. The adhesion of cells lacking actin filaments and actin-induced spreading was governed by surface interactions between the cell membrane and PLL alone. Compared with untreated Jurkat cells, the cells treated with latrunculin exhibited a significant reduction in adhesion area sizes (Figure 3A,B). Therefore, the adhesion and spreading of Jurkat cells on PLL were likely mediated by both surface interactions and actin polymerization at the leading edge. The average adhesion area size of cells that incorporated chol-gelatin was similar to that of cells treated with latrunculin B, suggesting that the incorporation of chol-gelatin into cell membranes suppresses the effect of actin polymerization-based cell spreading (Figure 3B). In addition, incorporation of cholgelatin into cells that were pretreated with latrunculin B resulted in only a slight decrease in the adhesion area size (Figure 3B), demonstrating that gelatins did not significantly weaken the interaction between the cell membrane and the PLL surface. Conversely, such molecular affinity did not appear to enhance the collective surface interactions between the cell membrane and the PLL-coated surface, either. This is contrary to the observation that soluble gelatin molecules bound to the PLL-coated surface nonspecifically (data not shown), although free gelatins in solution and membrane-bound gelatins may behave differently for the interaction with the PLL surface. These results suggest that chol-gelatin in the cell membrane does not significantly alter the surface adhesion force on PLL but perturbs actin polymerization-induced cell spreading. Next, we compared the dynamics of cell spreading under various conditions. Previous studies have shown that the early dynamics of cell spreading prior to actin polymerization-based spreading is governed by the viscosity of cell membranes.11 Our
scales (Figure S1B and Movie S1) and were clearly distinguishable from the adhered cells. In addition, cells adhered nonspecifically to defects in supported bilayers were more static and immobile than cells that adhered via chol-gelatins and thus were also visually distinguishable (data not shown). Rate of Intermembrane Bond Formation Increases with the Number of Free Cholesterol Moieties Displayed on Cell Membranes. We next examined whether the binding of cholesterol moieties to supported bilayers facilitated the cell−bilayer adhesions. Cells were preincubated with (chol)n-gelatins (n = 0.50 and 8.75) at various concentrations and then applied to supported bilayers after washing out excess chol-gelatins (Figure 2D,E). We found that (chol)8.75-gelatin (high cholesterol) consistently created more frequent cell adhesions than (chol)0.50-gelatin (low cholesterol). The number of adhesion events increased when the density of (chol)8.75gelatin in the cell membrane was higher, but such a trend was not observed for cells that incorporated (chol)0.50-gelatin (Figure 2D,E). These results demonstrated that the rate of the formation of cell−bilayer adhesions increased with the total number of free cholesterol moieties displayed on the cell membranes. Thus, the adhesion is most likely facilitated by the interactions between cholesterol and the supported bilayers. Increasing of the density of (chol)0.50-gelatin did not significantly enhance the rate of cell adhesion, probably because anchored (chol)0.50gelatin used one cholesterol for binding and there were fewer free cholesterols even with a larger number of total (chol)0.50gelatin molecules bound to the cell surface. Consequently, these cells had a much lower probability for triggering the adhesion, and we could resolve the differential effects of (chol)0.50-gelatin and (chol)8.75-gelatin in cell-bilayer adhesion (Figure 2D,E). As expected, in the absence of any chol-gelatin, Jurkat cells did not bind to bilayers (Figure 2D,E). The areas and diameters of adhesion sites were measured in RICM images (Figure 2F). Although the number of cholesterol moieties displayed on the cell surface varied under different conditions, we did not detect a significant difference in the size of adhesion sites. The adhesion area was unchanged in the range of 0.1−4.0 mg/mL for preincubation concentrations of both (chol)0.5-gelatin and (chol)8.75-gelatin, which corresponds to the maximum ∼15-fold (chol-gelatin) and ∼250-fold (total cholesterol moieties) differences in the average densities on cell membranes (Figure 1F,G). These results indicated that the cell−bilayer adhesion area did not spread with the increasing number of chol-gelatin and cholesterol moieties that may be located in the cell−bilayer interface and suggested a negative effect of chol-gelatin that suppresses cell spreading. The number of cell adhesion events was also significantly lower when the supported bilayers were preincubated with chol-gelatin (Figure 2E). These results were consistent with the observation in Figure 2B and again suggested that chol-gelatin in solution and chol-gelatin displayed on the cell surface may compete for interactions with the supported bilayers. This observation may be also related to the fact that direct cell−cell adhesion cannot be systematically induced in our current experimental conditions. To display a sufficient number of cholesterols on the cell surface to induce frequent cell adhesions, the cells needed to incorporate a substantial amount of chol-gelatin (Figure 2E). However, at such a high density of chol-gelatins, cell membranes may not be able to accommodate more cholesterol moieties displayed on other cells. Improving 1993
DOI: 10.1021/la504523c Langmuir 2015, 31, 1988−1998
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probably because of the different cell types. Representative time courses of images and adhesion diameters measured in the images showed rapid growth of the adhesion area in actindepolymerized cells, reaching a maximum area within 5−10 s (Figure 3C,D). Such rapid adhesion indicates the loss of viscosity in cell membranes by depolymerization of cortical actin filaments as previously shown.11 Chol-gelatin incorporation did not affect the dynamics of spontaneous adhesion of latrunculin B-treated cells on the PLL-coated surface (Figure 3C). In contrast, untreated Jurkat cells spread for ∼1000 s as also reported previously.11 Incorporation of chol-gelatin into untreated cells did not significantly alter the spreading dynamics but decreased the size of the adhesion area. These results suggested that chol-gelatin in cell membranes did not significantly alter the viscosity of the membrane structure, which governs the early dynamics of cell spreading.11 We found that the incorporation of chol-gelatin into cell membranes did not appear to alter the surface interaction force to PLL surface or the membrane viscosity that is modulated by cortical actin filaments but significantly suppressed cell spreading mediated by actively polymerizing actin filaments at the lamella. Deformation and spreading processes require energy for elastic bending and probably adjustment of osmolality. Such elastic energy loss is compensated by the gain in active actin polymerization, while chol-gelatin was most likely associated only with the outer leaflets of cell membranes and did not directly interact with or disturb cytoplasmic actin (Figure 1). Therefore, we hypothesized that one mechanism that could explain these observations may be mechanical regulation: the incorporated chol-gelatin might increase the rigidity of cell membranes and restrict cell deformation and spreading mediated by polymerized actins. At molecular and structural levels, this could be induced by bulky membranebound gelatin and/or membrane-inserted cholesterols. Such mechanical restriction could also suppress cell spreading on supported bilayers. Similar mechanical regulation of cell membranes has also been reported by incorporation of wheat germ agglutinin (WGA) into the membranes of red blood cells as directly demonstrated by a micropipet-based assay.42 Modulation of Cell Adhesion by Chol-Gelatin Is Not Cell Type Specific. If chol-gelatin modulates cell−bilayer adhesion though cholesterol−lipid bilayer interactions and membrane−actin, the observed phenomena should not be cell type specific. Therefore, we analyzed another cell line, drosophila S2. S2 cells are a semiadherent cell line of hematopoietic origin, which is known to form very large lamella, making the cell line a useful tool to study lamella formation.41 S2 cells also adhered to bilayers via chol-gelatin (Figure S1C), and chol-gelatin-incorporated S2 cells exhibited a smaller adhesion site area on PLL-coated surfaces (Figure S1D), demonstrating that chol-gelatin modulates adhesion of both Jurkat and S2 cells in a similar manner. S2 cells exhibited an even larger decrease in the adhesion area on PLL than Jurkat cells by incorporation of chol-gelatin (Figure S1E). This decrease occurred because many S2 cells form very large lamella even on PLL but such lamella formation was almost completely inhibited by incorporation of chol-gelatin (Figure S1D). These results suggest that the incorporation of cholgelatin into cell membranes also restricts actin-based spreading of S2 cells. Incorporation of a Physiological Adhesion Protein into Jurkat Cells Does Not Restrict Cell Spreading. Next,
Figure 3. (A) RICM images of Jurkat cells incubated on a PLL-coated glass surface for 1000 s. Untreated cells (left), cells pretreated with 4.0 mg/mL (chol)0.50-gelatin for 1 h, and cells pretreated with 20 μM latrunculin B for 1 h (right). (B) Average diameter of adhesion sites relative to the diameter of the whole cell. Cells that incorporated cholgelatin at various concentrations and/or were treated with 20 μM latrunculin B were plated on the PLL-coated surface and incubated for 1000 s. Error bars represent the SEM. (C) Representative time course of RICM images of cells spreading on the PLL-coated surface. Control Jurkat cells (untreated) were compared with cells that incorporated chol-gelatin and/or were treated with latrunculin B. (D) Representative plots for relative diameters of adhesion sites in cells as calculated from RICM images. Scale bars represent 5 μm.
observations were largely consistent with this finding and other previous studies except for some quantitative differences, 1994
DOI: 10.1021/la504523c Langmuir 2015, 31, 1988−1998
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Figure 4. (A) JCaM2 cells expressing CD2EM conjugated to mCherry were adhered to supported bilayers containing Alexa 647-labeled CD58. In the images, there are two cells with different expression levels of CD2, which exhibited very different adhesion areas as shown by the fluorescence of CD2 and CD58. (B) Normalized area of adhesion and the integrated fluorescence intensity of CD2 in images were plotted for more than 20 cells in the same bilayer sample. Average adhesion area of Jurkat cells mediated by (chol)8.75-gelatin (0.036) and the average size of the Jurkat cell cross section (1.0) are also plotted.
lation of cytoplasmic actin filaments. However, chol-gelatin molecules resist such cell deformation. Note that the full-length CD2 containing the cytoplasmic domain functions to actively induce actin polymerization and further spreading of Jurkat cells.26 Mechanisms of Chol-Gelatin-Mediated Cell Adhesion and Insights into the Design of Other Synthetic Adhesive Molecules. Schematics explaining our observations and models are shown in Figure 5. Chol-gelatin binds to the cell membrane most likely via a single cholesterol moiety, regardless of the number of cholesterol moieties per molecule. However, the structural basis for restriction of multiple anchor formation is not fully resolved. By having multiple cholesterols per gelatin molecule, the total number of cholesterol moieties displayed on the cell surface increases and the probability of the formation of cell-bilayer adhesion also increases (Figure 5A). However, cells do not spread, even with increasing numbers of cholesterols displayed on the cell surface. In contrast, the physiological adhesion protein CD2 induces cell spreading by having more molecular binding even in the absence of the connection to actin filaments. Note the binding affinity of CD2 for its ligand CD58 is as weak as that of chol-gelatin for cell membranes (Figure 5B). In experiments using the PLL-coated surface, we again found that incorporation of chol-gelatin suppresses cell spreading (Figure 5C). On PLL, cells spread by surface interaction and actin polymerization at the leading edges. Thus, after depolymerization of actin filaments, the surface forces primarily governed the areas of adhesion sites. However, the incorporation of chol-gelatin in cell membranes did not alter the area. In addition, the early dynamics of spreading, which was largely governed by cortical actin filaments, remained unchanged (Figure 5C). As one theory to explain our experimental data, we hypothesize that chol-gelatin incorporated in cell membranes enhances the rigidity of the cell membrane and suppresses
we compared adhesion mediated by chol-gelatin and physiological adhesion proteins. We examined adhesion between the CD2 receptor in Jurkat cell membranes and its ligand CD58 that was anchored to supported lipid bilayers. CD58 is expressed in B cells and dendritic cells and regulates the adhesion and signaling of T cells via the CD2−CD58 interaction. Interestingly, the affinity of the CD2−CD58 interaction measured in vitro is weak in solution (KD ∼ 9−22 μM and koff ∼ 4 s−1),43 which is comparable to the affinity of chol-gelatin for cell membranes (Figure 1). Such a weak interaction of CD2−CD58 has been suggested to be useful to allow both cell adhesion and deadhesion.44 We expressed the extracellular and transmembrane domains of CD2 (CD2EM) conjugated to mCherry in Jurkat cells to decouple the CD2−CD58 interaction and cellular actin network through the cytoplasmic domain of CD2, similarly to chol-gelatin in cell membranes, which did not link to cytoplasmic components. Because Jurkat cells express endogenous CD2, we used a CD2-deficient variant of Jurkat cells, JCaM2 cells. As reported previously, mobile CD58 molecules on supported bilayers created adhesion sites with Jurkat cells that were enriched with both CD2EM and CD58 (Figure 4A).26 We quantified CD2EM at the adhesion sites by the fluorescence intensity and plotted the values with the area of adhesion sites. A near linear relationship was found between the adhesion area and total CD2EM over several orders of magnitude (Figure 4B). As expected, cells that formed a larger adhesion area expressed a higher level of total CD2EM as indicated by fluorescence microscopy (data not shown). Thus, in contrast to chol-gelatin, CD2EM-CD58 binding induced cell spreading and deformed Jurkat cells, while the density of CD2EM-CD58 at the adhesion site was nearly constant as suggested by the linear relationship in Figure 4B. Therefore, Jurkat cells can spread by having more receptor-ligand pairs at membrane−membrane junctions without any direct modu1995
DOI: 10.1021/la504523c Langmuir 2015, 31, 1988−1998
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Figure 5. Schematics for the mechanisms of cell adhesion on supported bilayers mediated by chol-gelatin. (A) Conjugation of multiple cholesterols to one molecule does not improve the affinity of the gelatin−bilayer interaction because they bind via a single cholesterol moiety but improves cell− bilayer adhesion by displaying more cholesterol moieties on the cell surface. (B) Binding of CD2−CD58, but not chol-gelatin, induces cell deformation, even with increased numbers of chol-gelatin molecules. (C) Cell adhesion and spreading of Jurkat cells involve both membrane− surface interactions and active spreading by polymerized actin filaments at the leading edge. By incorporating chol-gelatin into cell membranes, actinbased deformation is restricted by enhancing membrane rigidity. By depolymerizing actin filaments, such deformation is also lacking. The viscosity that was regulated by cortical actin filaments governed spreading dynamics.
involved in cell spreading, although in general we believe that cell spreading is not favored in the context of osmolality. When cells are forced to deform, energy is required to adjust the osmolality. All these questions can be further investigated by testing other molecules as adhesive reagents. If the manners of adhesion by chol-gelatin reflect the properties of the two parts of the hybrid molecule, cholesterol and gelatin, shuffling different combinations of the components may provide a panel of novel adhesion molecules. For example, gelatin can be switched to more flexible polymer or natural membrane molecule, including CD2. Cholesterol can be replaced with a simple hydrocarbon chain with different length to avoid potential condensing effect. In addition, the control of the modification sites in backbone molecules can be useful. Membrane association of backbone molecules should be improved, when two anchors are located in close proximity, as seen in many physiological membrane-anchored proteins (e.g., Src, Ras, or Rab family proteins, or Gα proteins35). These investigations could directly lead to the development of different types of useful, synthetic adhesive reagents, including ones that are optimized to regulate direct cell−cell adhesion.
actin-polymerization based cell spreading. Such mechanical modulation was demonstrated for WGA binding.41 WGA is also not a physiological membrane-associated molecule. In contrast, such mechanical regulation was clearly absent for the physiological membrane molecule CD2 (Figures 4 and 5B). Thus, it may be important to know the structure of the membrane-bound gelatin to understand whether or how such mechanical regulation could be exerted on cell membranes. Furthermore, the inserted cholesterol moieties alone could contribute to membrane mechanics through the membrane condensing effect,37 or alternatively the combination of cholesterol insertion and gelatin binding could modulate the membrane mechanics. We could not rule out the effect of a membrane surface interaction force. However, in most experimental conditions tested, we believe the modulation of a surface force has only a minor role in the chol-gelatin mediated suppression of cell spreading. Other factors may also affect cell spreading. Cholgelatin was highly diffusive in lipid bilayers, and its diffusion coefficient was comparable to that of a single phospholipid in lipid bilayers, as shown by FRAP (fluorescent recovery after photobleaching) using supported bilayers (unpublished data).39 However, the mobility and distribution of chol-gelatin in cell membranes have not yet been fully examined. Cell adhesion could be affected if local condensing, depletion, or partial gelatin of chol-gelatin occurs. Osmolality may be another factor 1996
DOI: 10.1021/la504523c Langmuir 2015, 31, 1988−1998
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CONCLUSION We investigated a semisynthetic adhesive molecule, cholesterolmodified gelatin, and demonstrated our simple strategy of connecting two membranes via the multiple cholesterols on this molecule. We showed that the rate of cell adhesion to supported bilayers increases by incorporating and displaying more free cholesterols on the surface, while further cell spreading was restricted. Our study might provide a basis for the design and characterization of such hybrid molecules that may be potentially useful for modulation of cell adhesion not only in vitro but also in vivo.
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ASSOCIATED CONTENT
S Supporting Information *
Figure S1 and Movie S1. 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]; Ph +81-29-8592342 (Y.K.). Author Contributions
A.U. and M.O. contributed equally. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by an intramural grant of NIMS. We acknowledge the use of the soft material line facility at NIMS for experiments and thank all staff members of the facility, particularly R. Nagano and H. Morita for their help. We thank S. Hiromoto and T. Yamazaki for helpful discussions.
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