In eukaryotic cells, major biological processes take place in membranes and it is now recognized that molecular diversity and local non-equilibrium effects generate a high level of membrane heterogeneities. Our primary aim has been to study this spatio-temporal complexity and its role in the signal transduction processes underlying the T cell activation by combining advanced biophotonic observations on living cells with biological manipulations.
Using an original Fluorescence Correlation Spectroscopy (FCS) method we identified in the plasma membrane two major lateral confining forces that are lipid-dependent nanodomains and actin cytoskeleton meshwork. By characterizing lipid nanodomains in details we provided the first unambiguous evidence for the existence of membrane rafts in living cells. Through direct monitoring and controlled alterations of rafts in living cells, we then demonstrate that raft nanodomains are critically involved in the activation of different signaling pathways that are essential for cell physiology.
At the meant time, we have pursued investigating new biophotonics approaches following two axes: (1) by implementing nanoscopy imaging setup based on single molecule detection and reliable analytical methods and (2) by exploring new contrasting methods based on non-linear optics and high reolution wavefront sensor in tight collaboration with the group of Rigneault (Fresnel Institute) and companies (MaunaKea Technology & Phasics SA).
Our major goal is to progress one step further in our understanding of the role of membrane lateral dynamics and organization in T lymphocyte signaling, by analyzing the molecular interaction/association events at high spatial-temporal resolutions. A special emphasize is made at examination of the molecular dynamics in the plasma membrane to initiate and to integrate extracellular stimuli.
This progress of knowledge is crucially dependent upon reliable analytical methods based on the combination of single molecular sensitive detection approaches such as fluorescence correlation spectroscopy (FCS) and derivatives, of single particle tracking with optical tools allowing to manipulate the biological samples such as dynamic holographic optical tweezers.
1- Nanoscale lateral organization of the plasma membrane by svFCS
Since more than 50 years, the cell membrane has been a major research focus among biologists, chemists and physicists. Singer and Nicolson formulated in 1972 the fluid mosaic model proposing that the biological membranes can be considered as a two-dimensional liquid where all lipid and protein molecules diffuse laterally in a relatively free manner. This fluid mosaic model has been the prevailing view for many years, and even still being the case for some authors, but accumulating evidence over the past years has indicated that cell membranes are more “mosaic” than “fluid” – they constitute a laterally heterogeneous organization. However, this inhomogeneity is at present poorly determined, especially at the sub-micron and sub-second spatio-temporal length scales. In addition, factors underlying this inhomogeneity also are poorly characterized.
To investigate the dynamic and complex membrane lateral organization in living cells, we have developed an original approach based on molecule diffusion measurements performed by spot variation fluorescence correlation spectroscopy (svFCS). Our approach which is non invasive for the biological samples enables probing spatiotemporal membrane heterogeneities on living cells. Indeed, we have shown in a variety of cell types that both actin cytoskeleton meshwork and lipid-based nanodomains are instrumental for cell membrane compartmentalization: proteins and lipids can be dynamically confined within meshes or isolated domains at a suboptical spatial length and with a time lag of tens to hundreds milliseconds. When both the lipid-based nanodomains and actin cytoskeleton were disrupted, a free-like diffusion was observed for most of the examined lipids and proteins.
[[wysiwyg_imageupload::]]Trajectories of EGF receptors tagged by quantum dots on a COS-7 fibroblast, monitored by video-microscopy (at 36 ms per frame for 10 s) and localized in 3D, using astigmatic optics, by the Multi-Target Tracing home-made software. Color corresponds to z position, from blue (basal membrane) to red (lateral membrane and ruffles – apical membrane being out of focus).
2- Raft nanodomains play important role in cell signaling
Lipid rafts have been proposed to be involved in various cell functions, including in particular the signal transduction, with T lymphocytes being extensively studied in this context. Our ability to monitor directly dynamics and to induce accurate alterations of raft nanodomains in living cells have given us the unique opportunity to investigate adequately the functional role of rafts in cell signaling.
Activation of the PI3K/Akt signaling pathway, in response to external cell stimuli, triggers a set of events leading to cell growth, cell cycle entry, cell migration, and cell survival. This pathway is also essential in T cell activation. We have established a critical role for these nanodomains in activation of PI3K/Akt-dependent signaling pathway.
We observed that nanodomains are crucial for efficient membrane recruitment and phosphorylation of Akt upon formation of phosphatidylinositol-3,4,5 triphosphate in the inner-leaflet of the plasma membrane.
Fas-triggered cell death signaling is involved in the physio-pathology of both immune and non-immune cells. In collaborative studies with the group of A.-O. Hueber (CNRS UMR6543, Nice), we have shown that both Fas and FasL constitutively partition into raft nanodomains. These partitioning are required for activation of cell death signaling cascades upon Fas engagement by FasL. We have further demonstrated that Fas undergoes palmitoylation, which was found to be necessary for its raft association and cell death signaling.
3- New biophotonic instruments and analytical approaches
Single Particle Tracking (SPT) has been initially performed using submicron colloids coupled to membrane components by antibodies and observed by videomicroscopy. Reconstructing the trajectories provided, once again, an access to parameters such as the diffusion coefficient, by using analytical tools such as the Mean square Displacement (MSD). Further improvements came from the possibility to follow a single molecule, coupled to a single fluorophore. Another improvement was brought by the use of quantum-dots allowing over passing signal to noise ratio (SNR) and lifetime limitations of conventional dyes.
In order to provide a global view on the whole cell plasma membrane with ultra spatial resolution, we aimed at tracking single fluorescent molecule motion at high density of particles. Therefore, we have implemented a new robust algorithm namely “Multi Target Tracing” (MTT), this software is downloadable here, allowing us to achieve an exhaustive detection of the fluorescent molecules, to reconnect accurately the trajectories and to translate the data into a map of the local molecular dynamics. The ability to monitor the traces of a high number of targets allowed us to establish a sufficiently documented cartography of membrane dynamics, with suitable spatiotemporal resolution for the study of transient confinement his temporal analysis simultaneously revealed stable and evanescent confined area at the surface of living cells.
Since 2008, we have developed a new activity in the field of Raman imaging and cancer diagnosis. We are leading the CARS Explorer project, a European network funded through the FP7-HEALTH program. We aim at developing an endoscope based on non-linear optics and laser pulse phase shaping for functional in situ imaging in life science and biomedical research.
The adaptive immune response to pathogen invasion requires the stimulation of lymphocytes by antigen-presenting cells. We hypothesized that investigating the dynamics of the T lymphocyte activation by monitoring intracellular calcium fluctuations might help explain the high specificity and selectivity of this phenomenon. However, the quantitative and exhaustive analysis of calcium fluctuations by video microscopy in the context of cell-to-cell contact is a tough challenge. To tackle this, we developed a complete solution named MAAACS (Methods for Automated and Accurate Analysis of Cell Signals), in order to automate the detection, cell tracking, raw data ordering and analysis of calcium signals. By the use of MAAACS we evidenced that, when in contact with antigen-presenting cells, TCR engagement generates oscillating calcium signals and not a massive and sustained calcium response as was originally thought. We anticipate our approach providing many more new insights into the molecular mechanisms triggering adaptive immunity.
This software is downloadable here.