From analog to digital or how the membrane of T lymphocytes contributes to convert a signal into an appropriate biological response

During its lifetime a cell receives a multitude of signals from its environment. These signals are picked up by receptors on the surface. After contact with specific antigen (signal), the receptors for antigen of T cells are activated, integrate information received and convert the relative intensity of the association between the antigen and the receptor into a cellular response appropriate to the biological situation: proliferate, differentiate, migrate or die.

The T cell receptor is the centerpiece detecting the presence of foreign antigens. However, it is more or less in permanent contact with other molecular partners present at the membrane or in the vicinity thereof: enzymes such as tyrosine kinases LCK, ZAP 70, "biological switches" as RAS proteins. It is these partners that direct the cells’ response.
The membrane lipids are another key component of this machinery. Indeed, they can promote or prevent the formation of complex molecular signaling: they contribute to this effect to organize the architecture of the membrane.

What are the dynamics of this supramolecular assembly? In other words, on what spatial and temporal scales do the membrane components organize themselves? What are the control elements of this organization? What is the impact of these spatiotemporal scales and control elements on decoding the signals into a wide range of biological effects?


For a long time the membrane surrounding the cells was seen as a simple envelope in which swim the proteins that regulate the exchange of information between the outside and inside the cell.

At first, scientists explored this membrane with biochemical methods to isolate and identify the major constituents. Using detergents, they then learned to solubilize membrane in more gentle ways, revealing the extraordinary complexity of possible interactions between the protein constituents of the membrane. It was only recently that lipids, by a great diversity of structure, have emerged as regulatory elements of these interactions can generate the complex heterogeneous environment.

More recently, the membrane appeared "alive": its constituents are moving constantly in a subtle interplay of chemical equilibria: some molecules are separated, others interact, allowing the final assembly of molecular complexes and efficiently retain the necessary plasticity to adapt to life.

"These mechanisms are at play in less
than a second in a span of just a few tens of nanometers"

All these phenomena occur on extremely limited scales of time and space: the process that leads to the activation of T cell receptor takes several hundred milliseconds and the field observation measures a mere ten nanometers. To access this universe, the team was one of the world's first to combine in vitro reconstitution, in vivo observation and simulation.

"From a technical point of view, this universe is very difficult to reconstitute in vitro" says Didier Marguet. "Consequently, we attempted to analyze the events unfolding in real time in the plasma membrane of living cells. Using a novel approach of microscopy-based fluorescence correlation spectroscopy, we were able to describe the dynamics of the architecture of the membrane."

The multiple-target tracing (MTT) tool takes advantage of the high resolution provided by single-molecule sensitivity. It generates dynamic maps at high densities of tracked particles, thereby providing a global representation of molecular dynamics in cell membranes.
We illustrate the strength of MTT by analyzing the membrane dynamics of the epidermal growth factor receptor (EGFR) naturally expressed in COS-7 cells. The EGFR is fluorescently detected at the plasma membrane of live cells by immunostaining with quantum dots. This allowed the realization of snapshots and movies of cell membrane organization over a short period of time (~10 s). Copyright Sergé A, Bertaux N, Rigneaux H, Marguet D, CIML.

The FCS is a method of analysis that very sensitive to time and it approaches the condition of observations in individual molecules. In this case, the originality lies in obtaining spatial information in parallel with the analysis of temporal observations to distinguish among molecules in motion, those who spread freely, those remaining constrained in areas of confinement in the membrane. The data gathered from these studies have validated a long-debated assumption among biologists: the existence of "lipid rafts". Thanks to the rigidity conferred by their high content of sphingolipids and cholesterol, these nanodomains float in the lipid bilayer, thereby contributing to the recruitment and activation of different membrane proteins.

"We identified two motors that regulate
the dynamics of T cell membrane"

"The only measure of the lateral dispersion of the molecules does not reflect the mechanisms at work within the plasma membrane, much less that of a T lymphocyte! To ensure the robustness, speed and specificity of the response to antigen, the various partners in the cell membrane enter into a dialogue in two dimentions but also three dimentions with proteins localized in the membrane and molecules with which "swim" in the cytosol of the cell " says Hai Tao He.

"To refine our observations and understanding of all aspects of the model we have expanded our scope of observation to include mechanisms to slow the association of different partners and conduct modeling to identify relevant features describing the membrane" Didier Marguet reflects.

"We then identified two motors that regulate the confinement of molecules within the plasma membrane of T cells: one directly related to lipid nanodomains and second, non-exclusive with the precedent, located directly under the membrane and associated with the cytoskeleton protein actin, which gives the cell a large part of its mechanical properties. These two forces contribute to the establishment of an extremely accurate and effective zoning. In less than a second, all players are linked: the switch receives the analog signal coming from the receiver that has just bound to the antigen, the interpreter with the help of these partners in the membrane generates in turn the digital signal to be transmitted to the nucleus of the cell."

At these scales of time and space, life of the membrane, far from being fixed, is marked by a succession of "lipid dependent" phases governing meetings between the partners involved in the signaling of T cells. By developing new approaches to biophotonics, the objective of the team is now observing the life cycle of each molecule at this organizational nanoscale.