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 mu-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.
INTRODUCTION
The full understanding of the mechanism of the signal transduction mediated by the G-protein-coupled receptors still requires the unraveling of the dynamic organization of these multimolecular systems in cell membranes. To date, a large amount of information has been published regarding this question and evidence has now been accumulated for nonrandom distribution and collision of the receptors, protein G, and effectors. A compartmentation of the receptors and/or the other partners was suggested as accounting for the rapidity and specificity of signaling (Neubig, 1994; Ostrom et al., 2000), but experimental studies devoted to the question of the membrane organization and dynamics of the components of this signal transduction pathway are still very scarce.
Recently, the single particle tracking (SPT) technique was developed allowing observation of the movements of individual membrane proteins or lipids at the cell surface with nanometer spatial resolution (Saxton and Jacobson, 1997). A submicrometer particle (colloidal gold or fluorescent latex) is specifically attached to the molecule of interest. The displacement of the particle recorded by videomicroscopy exhibits the movement of the labeled molecule. Careful analysis of the trajectories is required to distinguish between the possible different modes of motion and can reveal submicroscopic or larger membrane structures. This method is certainly the most promising to address the question of the mechanism of membrane-associated functions (Jacobson et al., 1995; Cherry et al., 1998).
In the present study we address the question of the lateral diffusion of the receptor as part of the mechanism of the signal transduction mediated by G-protein-coupled receptors. The principal objectives of this work were, first, to characterize the movements of the receptor and identify an eventual compartmentation, and second, to give a simple physical interpretation for the observed behaviors leading to a consistent model for the membrane organization around the receptors. We chose to study the ji-opioid receptor, target of many analgesic drugs including opiates, as a complementary approach to the efforts in our laboratory directed to the global understanding of the signaling mechanisms of this receptor (Capeyrou et al., 1997; Lagane et al., 2000). Experiments were carried out on a ,cc-opioid receptor tagged with a T7 phage capsid protein at its amino-terminal extracellular domain allowing for the labeling by 40 nm gold colloids bearing T7-tag antibodies. The tagged receptors were tracked at the surface of normal rat kidney (NRK) fibroblast cells, chosen for their morphological characteristics suitable for single particle tracking experiments, where they were stably expressed and functional. We followed the movements of the receptors during 2 min with a 40 ms time resolution and a 15 nm spatial resolution. The mu-opioid receptors were found to exhibit two different classes of diffusion. A slow or directed diffusion mode and a mode superimposing a long-term random diffusion with a short-term confined diffusion consisting of a diffusion confined within a domain that itself diffuses, what we call the walking confined diffusion. A thorough statistical analysis of the trajectories supports the image that the confinement seen here is of dynamical nature and does not involve the presence of fences as invoked in the membrane skeleton fence model (Kusumi et al., 1993).
CONCLUDING REMARKS
The most striking feature of our results is the demonstration that the diffusion of a membrane receptor can be confined inside a domain due to the existence of interactions between proteins in the absence of membranous or extramembranous fences. We were able to draw such conclusions from the detailed statistical analysis we performed of our SPT data, demonstrating the interest of going beyond a "simple" fit of the MSD versus time plots. We emphasize here that MSD fits alone do not allow one to distinguish between physically different microscopic models for the diffusion. The model we propose here is consistent with both the long time behavior of the trajectories (MSD measurements) and with their short time statistics (local equilibriation histograms). Moreover the numerical values obtained for the various fitting parameters are related in a simple way, which is also explained by our model.
Let us mention here that the results found in this paper support the ideas of Abney and Scalettar (1995), where membrane organization and heterogeneity are brought about by interprotein interactions rather than an imposed compartmentalization. Finally, the relevance of our model based on experiments performed at 22 deg C is confirmed by the consistency found with the measurements done at 37 deg C. Thus, a challenging question will be now to identify among all the possibilities, which are the interactions dominating the system and what is responsible for their regulation.
The recently suggested compartmentation of the G-protein-coupled receptors is confirmed by our data that show a confined diffusion component to the behavior of the receptor. However, a great deal of work is still needed to be able to establish the functional implication of such a behavior. In particular, the effect of the binding of a ligand on the diffusion properties will be very informative and is currently under study in our laboratory.
We are grateful to J.F. Tocanne for having initiated this project and for stimulating discussions. We thank D. Choquet for helpful advice during the setting up of the SPT device. The stably transfected NRK-A cell line was established by M. Corbani and S. Ducasse.
This work was supported by the Nano-Objet Individuel program of the Centre National de la Recherche Scientifique and the Association pour la Recherche contre le Cancer.
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[Author Affiliation]
Frederic Daumas,* Nicolas Destainville,^ Claire Millot,* Andre Lopez,* David Dean,^ and Laurence Salome* *Institut de Pharmacologie et Biologie Structurale, CNRS UMR 5089, 205, route de Narbonne, 31077 Toulouse Cedex, France; and ^Laboratoire de Physique Quantique, IRSAMC, CNRS UMR 5626, 118, route de Narbonne, 31064 Toulouse Cedex, France
[Author Affiliation]
Submitted September 21, 2001, and accepted for publication August 19, 2002.
Address reprint requests to Dr. Laurence Salome, Institut de Pharmacologie et Biologie Structurale, CNRS UMR 5089, 205, route de Narbonne, 31077 Toulouse Cedex, France. Tel.: +33 (0)5 61 17 59 39; Fax: +33 (0)5 61 17 59 94; E-mail: laurence.salome@ipbs.fr.
