Protein overexpression can induce the elongation of cell membrane nanodomains.

In cell membranes, proteins and lipids are organized into submicrometric nanodomains of varying sizes, shapes, and compositions, performing specific functions. Despite their biological importance, the detailed morphology of these nanodomains remains unknown. Not only can they hardly be observed by conventional microscopy due to their small size, but there is no full consensus on the theoretical models to describe their structuring and their shapes. Here, we use a combination of analytical calculations and Monte Carlo simulations based upon a model coupling membrane composition and shape to show that increasing protein concentration leads to an elongation of membrane nanodomains. The results are corroborated by single-particle tracking measurements on HIV receptors, whose level of expression in the membrane of specifically designed living cells can be tuned. These findings highlight that protein abundance can modulate nanodomain shape and potentially their biological function. Beyond biomembranes, this mesopatterning mechanism is of relevance in several soft-matter systems because it relies on generic physical arguments.

Probing functionalized gold colloids for single particle tracking experiments.

Functionalized submicroscopic particles are currently used to label proteins or lipids at the surface of living cells for single particle tracking experiments. In many cases, it can be of crucial importance for the particle to be anchored to a single molecule. We have addressed this question for the labeling at the plasma membrane of NRK cells of the mu-opioid receptor bearing a T7 epitope at the N-terminus. Using biophysical methods we were able to prepare quasi-monovalent anti-T7 antibody conjugated gold colloids (40 nm diameter) leading to stable and specific binding to the receptor. The rational method, we report here, can be extended to design customized probes for the labeling of various tagged molecules.

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

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.