Monitoring receptor-ligand interactions between surfaces by thermal fluctuations

Abstract

We describe a new method for determining receptor-ligand association/dissociation events across the interface of two surfaces (two-dimensional binding) by monitoring abrupt decrease/resumption in thermal fluctuations of a biomembrane force probe. Our method has been validated by rigorous control experiments and kinetic experiments. We show that cellular on-rate of association can be measured by analysis of intervals from a dissociation event to the next association event (waiting times). Similarly, off-rate of molecular dissociation can be measured by analysis of intervals from an association event to the next dissociation event (bond lifetimes). Different types of molecular bonds could be distinguished by different levels of reduction in thermal fluctuations. This novel method provides a powerful tool to study cell adhesion and signaling mediated by single or multiple receptor-ligand species.

FIGURE 1

Thermal fluctuation method. (A) Photomicrograph of a BFP. A micropipette-aspirated RBC with a bead (probe) glued to its apex (left) was aligned against another bead (target) aspirated by another pipette (right). The right pipette was driven by a computer-programmed piezoelectric translator to move in a repeated approach-push-retract-hold-return test cycle. The left pipette was held stationary but the position of the probe was tracked by image analysis software to produce the data shown in panels B and C. (B and C) Horizontal position x of the right edge of the probe is plotted versus time t for a representative test cycle measuring the interaction between PSGL-1 coated on the probe and L-selectin (B) or P-selectin (C) coated on the target. Two periods of high positions in panel B are indicated by arrowheads. (D and E) Sliding standard deviations σ of 15 consecutive points of the position data in panels B and C, respectively. (F and G) Histograms of the σ-data in panels D and E (bars), respectively, each fitted by Eq. 1 (solid curves). Also superimposed on each panel are two histograms of σ-values calculated from x(t) data of two unencumbered probes recorded for the same duration of time (dotted curves). One unencumbered probe had the same spring constant of k = 0.15 pN/nm as the probe used to acquire the data in panels D and E. The other unencumbered probe had spring constant of k = 1.7 (F) or 0.8 (G) pN/nm. All histograms were normalized to have a unity area. The vertical dashed line σ_U = 3.8 nm on each panel is 1 SD (1.3 nm) to the left from the peak at 5.1 nm. The vertical solid line σ _L = 3.15 nm on each panel is 1.5 SD to the left from the same peak. These thresholds are marked in panels D and E as horizontal lines to identify bond association and dissociation events, which are marked by the respective down and up arrows. Arrowheads indicate intervals deemed indeterminate as to whether they corresponded to free or bound probes because data lay between the two thresholds. See also Supplementary Material, Supplementary Video.

FIGURE 2

Comparison between two methods for determining the presence of a bond. A total of 812 tests like those in Fig. 1 D for L-selectin-PSGL-1 interactions were segregated into two groups. The first group of 87 tests had σ-values immediately before the target return that were between the upper threshold σ_U = 3.8 nm and the lower threshold σ_L = 3.15 nm, which were deemed as indeterminate and excluded. The second group of 725 tests were further segregated into four subgroups depending on whether they had σ-values immediately before the target return above the upper threshold (no decreased fluctuation) or below the lower threshold (decreased fluctuation) and whether the returning target produced pulling or no pulling of the probe. The number of tests in each subgroup was plotted against the four conditions marked on the x-y plane (and also indicated on the top of each bar).

FIGURE 3

Exponential distributions of waiting times (A) and bond lifetimes (B). Pooled ensembles of 156 (L-selectin) or 190 (P-selectin) waiting times (A), defined as intervals from a dissociation event to the next association event, and 172 (L-selectin) or 240 (P-selectin) bond lifetimes (B), defined as intervals from an association event to the next dissociation event, of PSGL-1, respectively, interacting with L-selectin (▵) or P-selectin (○) were, respectively, sorted according to their durations. The natural log of the number of events with waiting times >t_w (A) or bond lifetimes >t_b (B) was, respectively, plotted against t_w or t_b, respectively, and fitted, respectively, by a straight line (not shown). The negative slopes of the best-fits represent cellular on-rate m_rm_lA_ck_on and off-rate k_off, respectively, whose values are indicated. The variations in these values are shown by the 95% confident intervals of the best-fit (lines). The goodness-of-fit was measured by the R² values, which are also indicated.

FIGURE 4

Kinetic parameters. Cellular on-rate (A) and off-rate (B) were plotted versus product of the site densities of the interacting molecules, L-selectin and PSGL-1. Data (points, error bar = 95% confident interval) were, respectively, fitted by a straight line that passed the origin (A) to estimate a molecular 2D effective on-rate 〈A_ck_on〉 (best-fit equation and R² were indicated) or by a horizontal line (B) to estimate the average off-rate 〈k_off〉 (indicated). (C) Comparison of kinetic rates of PSGL-1 interacting with L-selectin and P-selectin.

FIGURE 5

Comparison between theory and experiment. Frequencies of adhesion mediated by PSGL-1 interacting with L-selectin (▵) or P-selectin (○) were measured at indicated contact times (points, mean ± SE of three probe-target pairs) by averaging the adhesion scores (1 for pulling and 0 for no pulling at the end of the contact time of each test cycle) from 100 test cycles per probe-target pair. Theoretical adhesion frequencies as functions of contact time were predicted (curves) by Eq. 4 using the kinetic rates from Fig. 4 C and molecular densities measured from independent experiments (m_rm_l, = 0.2 and 0.15 × 10⁵ μm⁻⁴ for the L- and P-selectin cases, respectively).