Substrate-supported Planar Membranes Research Articles

Total Internal Reflection with Fluorescence Correlation Spectroscopy

The combination of total internal reflection illumination with fluorescence correlation spectroscopy (TIR-FCS) allows one to examine in quantitative detail a variety of biophysical properties related to the motions and interactions of fluorescent molecules near the interface of a transparent planar surface and an adjacent solution. Several experimental and theoretical aspects of this combination will be discussed. TIR-FCS has allowed characterization of local diffusion coefficients and concentrations of fluorescently labeled antibodies in solution but very close to substrate-supported phospholipid bilayers. TIR-FCS has also been used to examine the interaction kinetics of fluorescently labeled mouse IgG specifically and reversibly associating with the mouse receptor Fc-gamma-RII, which was purified and reconstituted into substrate-supported planar membranes. This method also has the potential, through the use of a single fluorescent reporter, of providing information about the thermodynamics/kinetics of nonfluorescent molecules which participate in surface binding mechanisms; e.g., those that compete with fluorescent reporters for surface-immobilized receptors or those that interact on the surface with the receptors and reduce or enhance the interaction of the fluorescent reporters with the surface binding sites.

Total internal reflection with fluorescence correlation spectroscopy: Applications to substrate-supported planar membranes.

In this paper, the conceptual basis and experimental design of total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is described. The few applications to date of TIR-FCS to supported membranes are discussed, in addition to a variety of applications not directly involving supported membranes. Methods related, but not technically equivalent, to TIR-FCS are also summarized. Future directions for TIR-FCS are outlined.

Total internal reflection with fluorescence correlation spectroscopy: Applications to substrate-supported planar membranes

Total internal reflection with fluorescence correlation spectroscopy: Applications to substrate-supported planar membranes

Ligand-Receptor Kinetics Measured by Total Internal Reflection with Fluorescence Correlation Spectroscopy

Total internal reflection excitation used in combination with fluorescence correlation spectroscopy (TIR-FCS) is a method for characterizing the dynamic behavior and absolute concentrations of fluorescent molecules near or at the interface of a planar substrate and a solution. In this work, we demonstrate for the first time the use of TIR-FCS for examining the interaction kinetics of fluorescent ligands in solution which specifically and reversibly associate with receptors in substrate-supported planar membranes. Fluorescence fluctuation autocorrelation functions were obtained for a fluorescently labeled IgG reversibly associating with the mouse receptor Fc γRII, which was purified and reconstituted into substrate-supported planar membranes. Data were obtained as a function of the IgG solution concentration, the Fc receptor surface density, the observation area size, and the incident intensity. Best fits of the autocorrelation functions to appropriate theoretical forms gave measures of the average surface density of bound IgG, the local solution concentration of IgG, the kinetic rate constant for surface dissociation, and the rate of diffusion through the depth of the evanescent field. The average number of observed fluorescent molecules, both in solution and bound to the surface, scaled with the solution concentration of IgG, observation area size, and Fc receptor surface density as expected. The dissociation rate constant and rate of diffusion through the evanescent field agree with previous results, and all measured parameters were independent of the incident intensity.

Rebinding of IgE Fabs at haptenated planar membranes: measurement by total internal reflection with fluorescence photobleaching recovery.

In previous work, a general analytical theory for ligand rebinding at cell surfaces was developed for a reversible bimolecular reaction between ligands in solution and receptors on a membrane surface [Lagerholm, B. C., and Thompson, N. L. (1998) Biophys. J. 74, 1215-1228]. This theory can be used to predict theoretical forms for data obtained by using total internal reflection with fluorescence photobleaching recovery (TIR-FPR) [Thompson, N. L., Burghardt, T. P., and Axelrod, D. (1981) Biophys. J. 33, 435-454]. Thus, one method by which the rebinding theory can be tested is to use TIR-FPR. In the work described herein, the reversible kinetics of mouse monoclonal anti-dinitrophenyl (DNP) IgE Fabs at substrate-supported planar membranes composed of 25 mol % DNP-conjugated phosphatidylethanolamine and 75 mol % dipalmitoylphosphatidylcholine have been examined by using TIR-FPR. Data were obtained as a function of the Fab solution concentration. Higher Fab concentrations reduce rebinding (and increase the fluorescence recovery rate) because different Fab molecules compete for the same surface-binding sites. Data were also obtained for solutions containing different volume fractions of glycerol. In these measurements, higher glycerol concentrations increase rebinding (and decrease the fluorescence recovery rate) because the solution viscosity is increased and the Fab diffusion coefficient in solution is decreased. The TIR-FPR data were quantitatively compared with theoretical predictions which follow from the general theory for rebinding at the membrane surface. The data were consistent with the theoretical predictions and, therefore, provide experimental verification of the previously developed theory.

High-Order Autocorrelation with Imaging Fluorescence Correlation Spectroscopy: Application to IgE on Supported Planar Membranes

The use of high-order autocorrelation with imaging fluorescence correlation spectroscopy is described. Fluorescently labeled, antitrinitrophenyl IgE antibodies were specifically bound to substrate-supported planar membranes composed of trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. The IgE-coated membranes were illuminated with a laser beam that was totally internally reflected at the substrate/solution interface. The evanescently excited fluorescence arising from the membrane-bound IgE was measured with a CCD camera. The images were corrected for background and for the elliptically Gaussian spatial dependence of the evanescent excitation intensity. A series of high-order pixel-to-pixel spatial fluorescence fluctuation autocorrelation functions was calculated from the images. The autocorrelation functions generated multiple independent parameters which were used to characterize the nonuniform spatial distributions of the membrane-bound IgE. These parameters varied with the IgE density and also changed significantly when the IgE-coated membranes were further treated with unlabeled, polyclonal anti-IgE. The high-order autocorrelation functions calculated from images of planar membranes containing fluorescently labeled lipids rather than bound, labeled IgE demonstrated that the spatial nonuniformities were prominent only in the presence of IgE. Images of fluorescent beads were used to demonstrate the principles and the methods.

Decreased IgG-FcγRII dissociation kinetics in the presence of a protein antigen

Total internal reflection fluorescence microscopy has been used to examine the interaction of a mouse monoclonal IgG2b, in the absence and presence of its protein antigen, with mouse FcγRII in substrate-supported planar membranes. Equilibrium association and kinetic dissociation constants were measured for the antibody S6-34.11, which is specific for bovine prothrombin fragment 1 (BF1). These measurements showed that BF1 induces a statistically significant decrease (30–40%) in the IgG-FcγRII dissociation kinetics. A corresponding increase in the equilibrium association constant was not observed, perhaps because the statistical accuracy of the equilibrium measurements is lower than that for the kinetic measurements. The consequences of these results for understanding the mechanism by which macrophages recognize and ingest opsonized targets are discussed.

Binding of the soluble, truncated form of an Fc receptor (mouse Fc gamma RII) to membrane-bound IgG as measured by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy has been used to investigate the binding of the soluble extracellular domain of mouse Fc gamma RII (sFc gamma RII) to an anti-trinitrophenyl monoclonal mouse IgG2b (GK14.1) specifically bound to substrate-supported planar membranes composed of dipalmitoylphosphatidylcholine (DPPC) and trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine (TNP-cap-DPPE). The equilibrium dissociation constants for sFc gamma RII at GK14.1-coated TNP-cap-DPPE/DPPC planar membranes containing 0.5-25 mol% TNP-cap-DPPE were approximately 1 microM. Total internal reflection with fluorescence photobleaching recovery was used to examine the dissociation kinetics. The fluorescence recovery curves were better described as a sum of two exponentials rather than by one exponential; the rates and fractional recoveries were approximately 1 s-1 (65%) and approximately 0.1 s-1 (35%). The similarity between the values of these equilibrium and kinetic parameters to those previously measured for the binding of IgG in solution to intact mouse Fc gamma RII reconstituted into planar membranes suggests that conformational changes which may occur when IgG is constrained to a membrane surface do not significantly affect the equilibrium or kinetics of IgG-mouse Fc gamma RII binding. The stoichiometry of sFc gamma RII-GK14.1 binding was 1:4, indicating that a significant fraction of the membrane-bound antibodies were not accessible for receptor binding. Possible mechanisms that might underlay the observed heterogeneity in sFc gamma RII-IgG binding kinetics are discussed.

Equilibrium, kinetics, diffusion and self-association of proteins at membrane surfaces: measurement by total internal reflection fluorescence microscopy.

The equilibrium, kinetics, diffusion and self-association of proteins at membrane/solution interfaces may deviate substantially from these processes in bulk solution. A set of methods for examining these phenomena combines substrate-supported planar model membranes and the use of evanescent illumination with laser-based, quantitative fluorescence microscopy. Measurement of the steady-state, surface-associated fluorescence can be used to examine the thermodynamic properties of proteins at membranes. When combined with fluorescence photobleaching recovery, this technique provides information about membrane-binding kinetics; and when combined with fluorescence pattern photobleaching recovery, measurement of the translational diffusion coefficients of proteins weakly bound to membranes is possible. The use of polarized evanescent illumination can provide information about the orientation distributions of adsorbed fluorophores. Fluorescence correlation spectroscopy provides information about the self-association (e.g. dimerization) of membrane-associated proteins.

Imaging fluorescence correlation spectroscopy: nonuniform IgE distributions on planar membranes

Fluorescence correlation spectroscopy is useful for detecting and characterizing molecular clusters that are smaller than or approximately equal to optical resolution in size. Here, we report the development of an approach in which the pixel-to-pixel fluorescence fluctuations from a single fluorescence image are spatially autocorrelated. In these measurements, tetramethylrhodamine-labeled, anti-trinitrophenyl IgE antibodies were specifically bound to substrate-supported planar membranes composed of trinitrophenyl-aminocaproyldipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. The antibody-coated membranes were illuminated with the evanescent field from a totally internally reflected laser beam, and the fluorescence arising from the IgE-coated membranes was recorded with a cooled CCD camera. The image was corrected for the elliptical Gaussian shape of the evanescent illumination after background subtraction. The spatial autocorrelation functions of the resulting images generated two useful parameters: the extrapolated initial values, which were related to the average cluster intensity and density; and the correlation distances, which were related to the average cluster size. These parameters varied with the IgE density, and unlabeled polyclonal anti-IgE enhanced the nonuniform IgE distributions. The autocorrelation functions calculated from images of planar membranes containing fluorescently labeled lipids rather than bound, labeled IgE demonstrated that the spatial nonuniformities were prominent only in the presence of IgE. Fluorescent beads were used to demonstrate the principles and the methods.

Dissociation Kinetics between a Mouse Fc Receptor (Fc.gamma.RII) and IgG: Measurement by Total Internal Reflection with Fluorescence Photobleaching Recovery

Total internal reflect with fluorescence photobleaching recovery (TIR-FPR) has been used to examine the dissociation kinetics between monomeric mouse IgG and a mouse Fc receptor (moFc gamma RII) reconstituted into substrate-supported planar membranes. IgG1, IgG2a, and IgG2b exhibited similar dissociation kinetics, whereas IgG3 did not bind. The fluorescence recovery curves for the IgG-moFc gamma RII interactions were best described by two reversible components (1.4 s-1, 66% and 0.06 s-1, 18%) and an irreversible component ( < 0.01 s-1, 16%). The kinetic parameters for a mouse anti-dinitrophenyl (DNP) IgG1 antibody were equivalent in the absence and presence of saturating amounts of DNP-glycine, demonstrating that possible allosteric changes which might occur in IgG1 upon hapten binding do not appreciably affect the kinetic characteristics of moFc gamma RII binding. The fluorescence recovery curves for polyclonal mouse IgG Fc were similar to those for intact IgG, showing that decreasing the size of the IgG 3-fold does not alter the dissociation rate. The dissociation kinetics of IgG1 decreased considerably in a low ionic strength buffer, indicating that the IgG1-moFc gamma RII interaction has significant electrostatic components.

Translational diffusion of bovine prothrombin fragment 1 weakly bound to supported planar membranes: measurement by total internal reflection with fluorescence pattern photobleaching recovery

Previous work has shown that bovine prothrombin fragment 1 binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca(2+)-specific manner. The apparent equilibrium dissociation constant is 1-15 microM, and the average membrane residency time is approximately 0.25 s-1. In the present work, fluorescence pattern photobleaching recovery with evanescent interference patterns (TIR-FPPR) has been used to measure the translational diffusion coefficients of the weakly bound fragment 1. The results show that the translational diffusion coefficients on fluid-like PS/PC planar membranes are on the order of 10(-9) cm2/s and are reduced when the fragment 1 surface density is increased. Control measurements were carried out for fragment 1 on solid-like PS/PC planar membranes. The dissociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also used to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diffusion coefficient was approximately 10(-8) cm2/s, which is consistent with that measured by conventional fluorescence pattern photobleaching recovery. This work represents the first measurement of a translational diffusion coefficient for a protein weakly bound to a membrane surface.

Total internal reflection fluorescence microscopy: application to substrate-supported planar membranes.

The use of total internal reflection illumination in fluorescence microscopy (TIRFM) is reviewed with emphasis on application to fluorescent macromolecules that specifically and reversibly bind to planar model membranes supported on glass or quartz substrates. Several methods for characterizing macromolecular motion and organization are discussed: the measurement of equilibrium binding curves to obtain values for equilibrium binding constants; the measurement of fluorescence photobleaching recovery curves to obtain values of kinetic rate constants and surface diffusion coefficients; and the measurement of fluorescence intensities as a function of the evanescent field polarization to characterize orientational order. Applications to cell-substrate contact regions are summarized and future directions of TIRFM are outlined.

Direct measurement of the weak interactions between a mouse Fc receptor (Fc gamma RII) and IgG1 in the absence and presence of hapten: a total internal reflection fluorescence microscopy study.

Total internal reflection fluorescence microscopy (TIRFM) has been used to directly measure the weak dissociation constants of IgG with a mouse IgG receptor (moFc gamma RII) that has been purified and reconstituted into substrate-supported planar membranes. Dissociation constants were measured for three different mouse monoclonal anti-dinitrophenyl (DNP) IgG1 antibodies and for polyclonal mouse IgG, in the absence and presence of saturating amounts of hapten (DNP-glycine). The dissociation constant for polyclonal mouse IgG was 3 microM, which agrees well with previous results. The dissociation constants for the three monoclonal antibodies with moFc gamma RII ranged from 2 microM to 3 microM and were not statistically different, suggesting that changes in moFc gamma RII dissociation constants which may exist within the IgG1 subclass are less than the error of the TIRFM measurements (approximately 20%). The measured IgG1-moFc gamma RII dissociation constants were not different for individual monoclonal antibodies in the absence or presence of saturating concentrations of DNP-glycine, directly showing that possible allosteric changes which might occur upon hapten binding and affect the equilibrium characteristics of Fc receptor binding are small. This work demonstrates a new approach for quantitatively examining the effects of solution components on weak receptor-ligand interactions.

Evidence from total internal reflection fluorescence microscopy for calcium-independent binding of prothrombin to negatively charged planar phospholipid membranes

Measurements to test for a proposed Ca2+-independent interaction of prothrombin with membranes containing acidic phospholipids are described. Fluorescein-labeled bovine prothrombin and its amino- and carboxy-terminal peptides, prothrombin fragment 1 and prethrombin 1, were added at various concentrations in the presence or absence of Ca2+ to the aqueous space bathing substrate-supported planar membranes composed of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), POPC/bovine brain phosphatidylserine (bovPS) (70:30 mol/mol), or POPC/1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) (70:30 mol/mol). Total internal reflection fluorescence microscopy (TIRFM) at the membrane-solution interface showed a significant enhancement by acidic lipids of prothrombin and prothrombin fragment 1 binding in the presence of 5 mM Ca2+, with apparent dissociation constants of 0.4 and 1 microM, respectively. TIRFM measurements indicated that bovPS and DOPG also significantly enhanced the binding of fluorescein-labeled prothrombin to the planar membranes in the absence of Ca2+, with apparent dissociation constants (13-30 microM) at least an order of magnitude larger than the Ca(2+)-dependent constant for prothrombin binding. Association of prethrombin 1 but not prothrombin fragment 1 with membranes in the absence of Ca2+ was enhanced by the presence of bovPS in the membranes, which suggests that the Ca(2+)-independent binding site(s) is (are) in the prethrombin 1 but not the fragment 1 portion of prothrombin.

Binding of IgG to MoFc.gamma.RII purified and reconstituted into supported planar membranes as measured by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.

Interaction of antibodies with Fc receptors in substrate-supported planar membranes measured by total internal reflection fluorescence microscopy

A procedure for constructing substrate-supported planar membranes using membrane fragments isolated from the macrophage-related cell line J774A.1 is described. Total internal reflection (TIR) fluorescence microscopy is employed to demonstrate that fluorescently labeled Fab fragments of a monoclonal antibody (2.4G2) with specificity for a murine macrophage cell-surface receptor for IgG (moFc gamma RII) bind to the planar model membranes. These measurements show that the planar membranes contain moFc gamma RII and yield a value for the association constant of 2.4G2 Fab fragments with moFc gamma RII equal to (9.6 +/- 0.4) x 10(8) M-1 and indicate that the surface density of reconstituted moFc gamma RII is approximately 50 molecules/microns 2. In addition, TIR fluorescence microscopy is used to investigate the Fc-mediated competition of unlabeled, polyclonal murine IgG with labeled 2.4G2 Fab fragments for moFc gamma RII in the planar membranes. These measurements indicate that the reconstituted moFc gamma RII recognized by 2.4G2 Fab fragments also retains the ability to bind murine IgG Fc regions and yield a value for the association constant of polyclonal murine IgG with moFc gamma RII equal to (1-5) x 10(5) M-1. This work represents one of the first applications of TIR fluorescence microscopy to specific ligand-receptor interactions.