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.

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

“…For instance, Kusumi's group determined that phospholipids undergo hop diffusion in cell membrane based on the control experiments that modified or depleted membrane skeleton (100). On the other hand, Salome's group believed that a G-protein-coupled receptor follows cage diffusion based on their visual inspection of the trajectories-only few hops from one domain to another were observed (69). With a close examination, our results resemble the hop diffusion described by Kusumi based on 40 nm gold particle tracking (Fig.…”

“…S8 for discussion on various types of confined diffusion). Although the exact expression of MSD depends on the shape of the confining region and on spatial dimensionality, the two-dimensional (2D) MSD of confined diffusion can be approximated by (39,69) MSD 2D ðtÞy L 2 3…”

“…where D micro is the short-term ''microscopic'' diffusion coefficient (39,69). This intradomain diffusivity D micro is measured using the same method as described in Brownian diffusion section.…”

“…Here we define the instantaneous diffusivity (D 0 ) as the linear MSD fitting result of each rolling window (regardless the classification result of that rolling window). Since MSD at a longer time lag is averaged over fewer samples (i.e., noisier), typically only the first 1-20 data points in the MSD curve are used for diffusivity estimation in a short time period (35,69,(71)(72)(73). The optimal number of data points used for D 0 estimation should be determined by expected diffusion coefficient, tracking error, size of time step, and track duration.…”

What do diffusion measurements tell us about membrane compartmentalisation? Emergence of the role of interprotein interactions

“…In the work performed on the μ opioid receptor, it was also observed that the macroscopic diffusion coefficient, D M , is correlated to L 2 : D M ∼ L 2 . The detection of jumps by a new algorithm (see "Appendix 1") showed that the trajectories do not display enough jumps over actinlike barriers to account for the observed relationship [48].An alternative mechanism known as "interacting Brownian particle" model was proposed to explain the observed diffusion behaviours [15]. It stated that attractive longrange interactions can be sufficiently strong to ensure the stability of small confined assemblies of proteins without invoking external barriers (see Fig.…”