Confined diffusion without fences of a g-protein-coupled receptor as revealed by single particle tracking

Abstract

Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the micro -opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients.

FIGURE 1

Video-enhanced differential interferential contrast images of gold colloids attached to the lamellipodial region of NRK-μ cells with the associated 2-min-long trajectories shown as an overlay (white line). Two types of movement were observed: (a) slow displacement and (b) apparently restricted rapid diffusion with the slow long-term component shown in black (see text). Below each image the corresponding mean-square displacement (MSD) versus time plot (gray line) is reported and its theoretical fit (black line) for the associated diffusion mode (c) slow or directed diffusion (see Eq. 2) and (d) walking confined diffusion (see Eq. 3).

FIGURE 2

MSD versus time plot at short time interval showing the determination of D_0-2, the microscopic diffusion coefficient. D_0-2 is calculated from the slope of the straight line connecting 0–2δt using MSD(t) = 4D_0-2t. For this experiment, D_0-2 = 9.3 × 10⁻¹⁰ cm²/s.

FIGURE 3

Plot of the microscopic diffusion coefficients D_0-2 against D_micro derived from the plot of the MSD versus time by the slope at the origin for D_0-2 and by the fit with the theoretical expressions for D_micro. The points are aligned on a straight line of slope 1 showing a good correlation between D_0-2 and D_micro for slow or directed diffusion (•) and walking confined diffusion (○).

FIGURE 4

Histogram showing the distribution of the microscopic diffusion coefficient D_0–2. The dashed line separates the populations found in slow or directed diffusion (black bars) and walking confined diffusion modes (white bars).

FIGURE 5

Histogram showing the distribution of the characteristic domain size L: the average value of L is 0.15 ± 0.10 μm.

FIGURE 6

Plots of the microscopic and macroscopic diffusion coefficients D_0-2 and D_MACRO against the characteristic domain size L. The two sets of points are both aligned on straight lines of slope 2 showing that D_0-2 ∼ L² and D_MACRO ∼ L².

FIGURE 7

Histograms of x(t + nδt) – x(t) for nδt between 0–1 s (black circles) and 0–4 s (gray circles) showing a Gaussian equilibrium distribution within the domain (Gaussian fits of the distributions are drawn in solid lines). One sees that there is no broadening of the Gaussian between 0–1 to 0–4 s, showing that the domain itself does not diffuse significantly on these time scales and that the system is equilibrated within the domain at timescales greater than 1 s.

FIGURE 8

Plot of D_0-2(5 s) as a function of L(5 s) extracted from a single trajectory over intervals of 5 s. The points are aligned on a straight line of slope 2 showing that D_0-2(5 s) ∼L²(5 s).