Didier Marguet

Homology between reaper and the cell death domains of Fas and TNFR1.

When engaged by antibodies or by their respective li-gands, the cell surface molecules Fas and TNFR1 (the 55 kDa tumor necrosis factor [TNF] receptor) can transduce into cells a signal that often leads to cell death (Armitage, 1994; Nagata and Golstein, 1995). Fas and TNFR1 have homologous extracellular cysteine-rich domains that make them members of the nerve growth factor receptor super-family. They are also homologous in their cytoplasmic regions for a stretch of 60-70 amino acids, called the death domain, which makes them different from most other members of this superfamily. This dual but dissociated homology, the autonomous death signaling ability of the fact that they are encoded within the last exon of their respective genes (Behrmann et al., 1994) suggest that Fas and TNFR1 may derive from a chimeric ancestor molecule. Such a molecule would have brought together an extracellular region, ancestor to a nerve growth factor receptor domain, and an intracellular cell death signaling module. We reasoned that a derivative of the latter might still exist as an autonomous entity and therefore looked for an intracellular molecule involved in cell death and of about the same size as the death domains. Reaper, discovered through an elegant genetic approach by White et al. (1994), is a 65 amino acid peptide, the expression of which is both necessary and sufficient for developmental cell death and at least some types of experimental cell death in Drosophila. Mere visual inspection showed homology between reaper and the death domains of TNFR1 and, to a lesser extent, of Fas (Figure 1). This homology was particularly significant because it involves Drosophila and mammalian molecules and because all of these are able to signal cell death. Closer inspection suggested some heterogeneity in the distribution of homologies along reaper. In particular, the middle third of reaper shows the highest density of homologies with TNFRI. Reaper is involved in a pathway signaling cell death in Drosophila (White et al., 1994). Expression of reaper is both necessary and sufficient to proceed to the next step of this pathway (White et al., 1994). The emergence during evolution of a chimeric molecule covalently linking reaper to an extracellular module would have allowed direct access from the cell exterior to this cell death signaling pathway. For instance, a cell death pathway would thereby have become directly accessible via Fas to cytotoxic T cells bearing the Fas ligand (Rouvier et al., 1993; Suda …

Glycosylation-Dependent IFN-γR Partitioning in Lipid and Actin Nanodomains Is Critical for JAK Activation

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling inxa0vivo. VIDEO ABSTRACT.

Probing the plasma membrane organization in living cells by spot variation fluorescence correlation spectroscopy.

While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells. Here, we describe a method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization. The method is based on questioning the relationship between the diffusion time τ(d) and the squared waist of observation w(2). Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations. These investigations have allowed significant progress in the characterization of cell membrane lateral organization at the suboptical level, and have provided, for instance, compelling evidence for the in vivo existence of raft nanodomains.

Barcoding T cell calcium response diversity with methods for automated and accurate analysis of cell signals (MAAACS).

We introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells.

Independent Synchronized Control and Visualization of Interactions between Living Cells and Organisms

To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints. Here, we have developed an original setup comprising two independent modules: holographic optical tweezers, which offer a versatile and precise way to move multiple objects simultaneously but independently, and a confocal microscope that provides fast three-dimensional image acquisition. The optical decoupling of these two modules through the same objective gives users the possibility to easily investigate very early steps in biological interactions. We illustrate the potential of this setup with an analysis of infection by the fungus Drechmeria coniospora of different developmental stages of Caenorhabditis elegans. This has allowed us to identify specific areas on the nematodes surface where fungal spores adhere preferentially. We also quantified this adhesion process for different mutant nematode strains, and thereby derive insights into the host factors that mediate fungal spore adhesion.

A user's guide for characterizing plasma membrane subdomains in living cells by spot variation fluorescence correlation spectroscopy

Due to the intrinsic molecular Brownian agitation within plasma membrane and the vast diversity of membrane components, it is expected that the plasma membrane organization is highly heterogeneous with the formation of local complex multicomponent assemblies of lipids and proteins on different time scales. Still, deciphering this lateral organization on living cells and on the appropriate length and temporal scales has been challenging but is crucial to advance our knowledge on the biological function of the plasma membrane. Among the methodological developments based on biophotonics, the spot variation FCS (svFCS), a fluorescent correlation spectroscopy (FCS)-based method, has allowed the significant progress in the characterization of cell membrane lateral organization at the suboptical level, including to providing compelling evidence for the in vivo existence of lipid-dependent nanodomains. The aim of this chapter is to serve as a guide for setting and applying the svFCS methodology to study the plasma membrane of both adherent and nonadherent cell types.

Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation

Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities. However, their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Here, we investigate their role, and in particular that of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in TCR PM dynamics and activity in a mouse T-cell hybridoma upon ectopic expression of a PM-localized inositol polyphosphate-5-phosphatase (Inp54p). We observed that dephosphorylation of PI(4,5)P2 by the phosphatase increased the TCR/CD3 complex PM lateral mobility prior stimulation. The constitutive and antigen-elicited CD3 phosphorylation as well as the antigen-stimulated early signaling pathways were all found to be significantly augmented in cells expressing the phosphatase. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, thus resulting in an increased CD3 availability for interactions with Lck kinase. This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Our data thus suggest that PIs play a key role in the regulation of the TCR/CD3 complex dynamics and activation at the PM.

A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses

Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power.

A Theoretical High-Density Nanoscopy Study Leads to the Design of UNLOC, a Parameter-free Algorithm

Single-molecule localization microscopy (SMLM) enables the production of high-resolution images by imaging spatially isolated fluorescent particles. Although challenging, the result of SMLM analysis lists the position of individual molecules, leading to a valuable quantification of the stoichiometry and spatial organization of molecular actors. Both the signal/noise ratio and the density (Dframe), i.e., the number of fluorescent particles per μm2 per frame, have previously been identified as determining factors for reaching a given SMLM precision. Establishing a comprehensive theoretical study relying on these two parameters is therefore of central interest to delineate the achievable limits for accurate SMLM observations. Our study reports that in absence of prior knowledge of the signal intensity α, the density effect on particle localization is more prominent than that anticipated from theoretical studies performed at known α. A first limit appears when, under a low-density hypothesis (i.e., one-Gaussian fitting hypothesis), any fluorescent particle distant by less than ∼600xa0nm from the particle of interest biases its localization. In fact, all particles should be accounted for, even those dimly fluorescent, to ascertain unbiased localization of any surrounding particles. Moreover, even under a high-density hypothesis (i.e., multi-Gaussian fitting hypothesis), a second limit arises because of the impossible distinction of particles located too closely. An increase in Dframe is thus likely to deteriorate the localization precision, the image reconstruction, and more generally the quantification accuracy. Our study firstly provides a density-signal/noise ratio space diagram for use as a guide in data recording toward reaching an achievable SMLM resolution. Additionally, it leads to the identification of the essential requirements for implementing UNLOC, a parameter-free and fast computing algorithm approaching the Cramér-Rao bound for particles at high-density per frame and without any prior knowledge of their intensity. UNLOC is available as an ImageJ plugin.