Measuring Receptor–Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods
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- Chen, W., Zarnitsyna, V.I., Sarangapani, K.K. et al. Cel. Mol. Bioeng. (2008) 1: 276. doi:10.1007/s12195-008-0024-8
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Interactions between surface-anchored receptors and ligands mediate cell–cell and cell–environment communications in many biological processes. Molecular interactions across two apposing cell membrane are governed by two-dimensional (2D) kinetics, which are physically distinct from and biologically more relevant than three-dimensional (3D) kinetics with at least one interacting molecular species in the fluid phase. Here we review two assays for measuring 2D binding kinetics: the adhesion frequency assay and the thermal fluctuation assay. The former measures the binding frequency as a function of contact duration and extracts the force-free 2D kinetics parameters by nonlinearly fitting the data with a probabilistic model. The latter detects bond formation/dissociation by monitoring the reduction/resumption of thermal fluctuations of a force sensor. Both assays are mechanically based and operate at the level of mostly single molecular interaction, which requires ultrasensitive force techniques. Characterization of one such technique, the biomembrane force probe, is presented.
KeywordsAdhesion frequency assayThermal fluctuation assayMicropipetteBiomembrane force probeKineticsReceptor–ligand interaction
Communication between cells with each other and with their environment is mediated via specific receptors on their surfaces. Although unraveled genome for key organisms has provided a lot of information about different surface molecules as corresponding gene products, only a fraction of them have functions assigned and even fewer have been studied in detail. Traditionally, receptor–ligand interactions are characterized by ensemble assays with at least one of the molecules purified from the cell membrane. The assays are carried out in the fluid phase, e.g., using the surface plasmon resonance technique. These are referred to as three-dimensional (3D) assays and are described in terms of a chemical reaction framework through receptor and ligand concentrations, kinetic rates, and binding affinity. While sufficient for a proper description in homogeneous solution, these properties by themselves are often insufficient for determining the interaction of the same molecules residing in their natural environment on the cell membrane. This is because cell surface molecules can be clustered, partitioned in cell surface structures such as membrane rafts, or linked to cytoskeleton directly or via other docking molecules, which can impact binding by signaling that changes molecular conformations. On the cell, molecules of interest may be abundant but inactive due to specific cellular environment or the same molecules may exhibit different activities in different areas or times. These features make ensemble assays insufficient, even if they are carried out on the cell surface, because the properties so measured represent ensemble averages only. It also calls for single molecule studies because these experiments probe molecular interactions one by one, thereby allowing for measurement of not only average properties but also their distributions.
Another important feature of receptor-mediated cell adhesion is that molecular interactions take place in two dimensions (2D) as both receptors and ligands are anchored to the respective surfaces of two apposing cells or a cell and a substrate. This situation is ideal for single-molecule force techniques because the sensitive force probes can be functionalized with interacting molecules. A molecular interaction manifests as a mechanical force through a receptor–ligand bond that physically connects two surfaces, one of which can be the force probe (the other is referred to as the target surface in this paper). Single-molecule biomechanical experiments with atomic force microscopy (AFM) or other ultrasensitive force techniques for measuring unbinding forces for dynamic force spectroscopy analysis or unfolding of protein domains are discussed in other papers of this thematic issue. In this review, we summarize our previous work that uses an adhesion frequency assay2 and a thermal fluctuation assay1 to extract kinetic information of receptor–ligand interaction from measured binding events.
Adhesion Frequency Assay
Unlike 3D assays where soluble molecules in the fluid phase can diffuse to the vicinity of the counter-molecules, 2D assay requires that the force probe and the target surface be brought into contact because interaction would not be physically possible if the two surfaces are separated by distances greater than the span of the receptor–ligand complex. Thus, the initiation of interaction is, in a sense, “staged” by the experimenter who puts the two surfaces into contact, controls the area and duration of the contact, and observe the outcome of such an adhesion test at the end of the contact when the two surfaces are separated.
To estimate Pa from the average adhesion score in a series of repeated contact–separation cycle (called adhesion test cycle) requires that the parameters on the right-hand side of Eq. (6) be constants. In the micropipette adhesion frequency assay,2 the adhesion test cycle is controlled via computer-driven micromanipulation to ensure each contact is as close to identical to any other contacts as possible to yield constant Ac and tc. The “apparent” contact area Ac* can be visualized microscopically, as shown in the side view photomicrograph of Fig. 1b. However, the functional contact area Ac depends on the microtopology of the cell surface.14 Furthermore, making a contact is a process that starts from a point to the final area, which requires time (termed lead time). Similarly, separating a finite contact and stretching the RBC membrane to the point when visual detection can be reliably made is also a process that requires time (termed dead time). It is implied in the derivation of Eq. (6) that the sum of these times is much smaller than tc such that the lead time and dead time are negligible during the course of a test cycle. This requirement places a limitation to the temporal resolution of the assay such that the half-life of the molecular interaction in question has to be much longer than the sum of lead time and dead time.2
The molecular densities mr and ml are usually assumed constant because the contact time tc (<1 min) is much shorter than the time td required to recruit molecules into the apparent contact area Ac* (∼3 μm2) from outside by diffusion. Indeed, for a typical diffusion coefficient D ∼ 0.01 μm2/s of cell surface molecules td can be estimated as Ac*/D ∼ 5 min.13 However, this assumption will be invalid if engagement of receptors by their adhesive ligands triggers cell signaling that results in reorganization of cell surface structures, such as changes in clustering of receptors and/or their partitioning in membrane microdomains. Signaling can also alter the kinetic rates and binding affinity, thereby violating the assumption of constant Ka, k−1, and/or k+1. These events can occur in short time, thereby complicating the analysis and interpretation of the adhesion score sequence measured from the adhesion frequency assay. In fact, a recent study has demonstrated that the adhesion probability of a test cycle in a repeated test series may depend on the outcome of the prior test(s) in the sequence.18 All three possible cases have been observed: the adhesion probability of the next test can be increased (positive memory), decreased (negative memory), or unchanged (no memory) by the adhesion of the immediate past test. The underlying causes of the memory effects are not clear, but may involve signaling that transiently changes the surface organization of the interacting molecules and/or their binding affinity.
Returning to the simple case with constant parameters, Eq. (6) states that the likelihood of observing adhesion, Pa, depends on the time when the observation is made relative to the time when the contact is initiated, i.e., the contact duration, tc. If the adhesion frequency is measured over a range of contact durations, fitting Eq. (6) to the measured Pa vs. tc binding curve then allows estimation of the 2D kinetic rates and binding affinity, provided that the receptor and ligand densities are measured from independent experiments.
Characterization of BFP
The thermal fluctuation assay, to be discussed in the next section, requires an ultrasensitive force sensor with a soft spring (the spring constant should be smaller than those of the interacting molecules), such as a BFP or optical tweezers. A BFP is a high-tech version of a micropipette, which includes a probe bead glued to the apex of the micropipette-aspirated RBC as a picoforce transducer (Fig. 3, left). Observed under an inverted microscope (Zeiss Axiovert 100 with 40× objective and 4× video tube), the position of the probe bead is tracked by a high-speed camera (1,500 frames per second, Cooke SensiCam) with customized and advanced image analysis software to achieve high spatial precision. In order to achieve high frame rates, the high-speed camera only captures a narrow strip of the field of view (1024 × 27 pixels), which is binned into a single line. This line is then transferred from the camera to the computer to carry out the online image analysis to determine the darkest position of the left edge of the probe bead (Fig. 3). A target bead (or cell) is aspirated by an apposing micropipette (Fig. 3, right) driven by a computer-controlled piezo translator (Physik Instrument).
Thermal Fluctuation Assay
The adhesion frequency assay discussed in section “Adhesion Frequency Assay” extracts kinetic information from the dependence of adhesion frequency Pa on contact duration tc.2 Adhesion is measured mechanically by separating the force probe (pressurized RBC or AFM cantilever) from the target to detect the presence of a receptor–ligand bond or bonds that connect the two surfaces at the end of a contact but not when a bond forms or dissociates during the contact. Therefore, kinetics of molecular interaction must be inferred from fitting a model, e.g., Eq. (6), to the Pa vs. tc data (Fig. 2).2 By comparison, a recently developed thermal fluctuation assay can pinpoint the association and dissociation events at the single-bond level during the contact period without separating the two surfaces.1 This greatly enhances the quantity, quality, and reliability of the information obtained, which makes kinetic measurements much simpler and more robust.
This difficulty can be overcome by using softer force probes with much smaller spring constants, such as those in optical tweezers or BFP.1 The basic experimental protocol consists of the following cycle. The piezo translator brings the receptor-coated target bead to briefly contact with the ligand-coated probe bead, retracts it to a desired position with sub-nanometer precision, holds it there for a given duration, and returns it to original position. This cycle is repeated many times to acquire an ensemble of data for statistical analysis as in the adhesion frequency assay. The position of the probe bead is continuously recorded and analyzed for detection of events of formation and dissociation of receptor–ligand bonds.1
Figure 8 is similar to Fig. 7; however, the much softer spring constant of the BFP compared to AFM resulted in clear changes in the displacement fluctuations when a bond is formed or dissociates (Figs. 8a, b compared to Figs. 7a, b), which manifests as a sudden decrease or increase in the standard deviations (Figs. 8c, d compared to Fig. 7c, d), enabling us to pinpoint the time when bond formation or dissociation occurs even when the force probe is placed so close to the target that bond dissociation does not result in a significant shift in the mean displacement (Fig. 8b compared to Fig. 8a and Figs. 7a, b). The two peaks in each of the standard deviation histograms corresponding to the free and bound force probe are clearly separated with minimal overlaps (Figs. 8e, f compared to Figs. 7e, f). The amount of left shift of the displacement standard deviation resulted from bond formation is smaller for P-selectin than for L-selectin, consistent with the fact that P-selectin has a smaller molecular spring constant than L-selectin. This suggests that the thermal fluctuation assay can distinct the type of bonds in addition to identifying the bond formation and dissociation events.
The validity of the thermal fluctuation assay can be tested by the sensitivity of the kinetic parameters estimated to the molecular interactions assayed, e.g., L-selectin vs. PSGL-1 and P-selectin vs. PSGL-1. Histograms of waiting times and lifetimes of these two interactions at comparable site densities are compared in Figs. 10a and 10b. It clearly shows that P-selectin has faster on-rate, but slower off-rate, to PSGL-1 than L-selectin. The kinetic rates are compared in Fig. 11c, which are consistent with previous finding that P-selectin8 has much higher affinity and slower off-rate to PSGL-1 than L-selectin.9
The thermal fluctuation method can be further validated by comparing the 2D kinetic rates measured by this method with those measured by the adhesion frequency assay,2 which has been extensively used to determine many receptor–ligand interactions. As described in the previous section, rather than measuring rupture forces, the adhesion frequency assay estimates the likelihood of adhesion, or adhesion probability, Pa, from the frequency of adhesion enumerated from a large number of repeated controlled contacts. Pa is related to the contact time tc through a probabilistic model described by Eq. (6) in adhesion frequency assay session.2
In this paper, we have reviewed two assays for measuring two-dimensional binding kinetics of a low number of receptor–ligand interactions. The 2D nature allows reaction kinetics to be assayed mechanically. That such binding is mediated by a low number of molecular interactions requires ultrasensitive force techniques and a probabilistic kinetic framework for analysis. These assays measure 2D force-free on- and off-rates. Off-rates of biomolecular interaction are known to be regulated by applied force,6,11 which is treated by other papers in this thematic issue. Two-dimensional kinetics of a large number of molecular interactions can also be assayed using a recently developed fluorescence-based assay.13,17 Collectively, these assays provide useful methodologies for studying molecular interactions across two apposing surfaces.
We thank our coworkers of references Chen et al.1 and Huang et al.5 who contributed the original data that are discussed here. This work was supported by National Institutes of Health Grants AI38282, AI44902, and HL091020. W.C. is a Predoctoral Fellowship recipient of the American Heart Association (Greater Southeast Affiliate).