François Amblard

Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity

Induction of immunity and peripheral tolerance requires contacts between antigen-bearing dendritic cells (DCs) and cognate T cells. Using real-time two-photon microscopy, we have analyzed the dynamics of CD8+ T cells in lymph nodes during the induction of antigen-specific immunity or tolerance. At 15–20 h after the induction of immunity, T cells stopped moving and established prolonged interactions with DCs. In tolerogenic conditions, despite effective initial T cell activation and proliferation, naive T cells remained motile and established serial brief contacts with multiple DCs. Thus, stable DC–T cell interactions occur during the induction of priming, whereas brief contacts may contribute to the induction of T cell tolerance.

Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells

The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.

Endocytosis is required for E-cadherin redistribution at mature adherens junctions

E-cadherin plays a key role at adherens junctions between epithelial cells, but the mechanisms controlling its assembly, maintenance, and dissociation from junctions remain poorly understood. In particular, it is not known to what extent the number of E-cadherins engaged at junctions is regulated by endocytosis, or by dissociation of adhesive bonds and redistribution within the membrane from a pool of diffusive cadherins. To determine whether cadherin levels at mature junctions are regulated by endocytosis or dissociation and membrane diffusion, the dynamics of E-cadherin were quantitatively analyzed by a new approach combining 2-photon fluorescence recovery after photobleaching (FRAP) and fast 3D wide-field fluorescence microscopy. Image analysis of fluorescence recovery indicates that most E-cadherin did not diffuse in the membrane along mature junctions, but followed a first order turn-over process that was rate-limited by endocytosis. In confluent cultures of MCF7 or MDCK cells, stably expressed EGFP-E-cadherin was rapidly recycled with spatially uniform kinetics (50 s in MCF7 and 4 min in MDCK). In addition, when endocytosis was pharmacologically blocked by dynasore or MiTMAB, no fluorescence recovery was observed, suggesting that no endocytosis-independent membrane redistribution was occurring. Our data show that membrane redistribution of E-cadherin molecules engaged in mature junctions requires endocytosis and subsequent exocytosis, and lead to the notion that E-cadherins engaged at junctions do not directly revert to free membrane diffusion. Our results point to the possibility that a direct mechanical coupling between endocytosis efficiency and cadherin-mediated forces at junctions could help to regulate intercellular adhesion and locally stabilize epithelia.

Molecular analysis of microscopic ezrin dynamics by two-photon FRAP

Ezrin plays a key role in coupling signal transduction to cortical cell organization. This actin–membrane linker undergoes a series of conformational changes that modulate its interactions with various partners and its localization in membrane or cytosolic pools. Its mobility and exchange rates within and between these two pools were assessed by two-photon fluorescence recovery after photobleaching in epithelial cell microvilli. Analysis of ezrin mutants with an altered actin-binding site revealed three ezrin membrane states of different mobilities and exchange properties, reflecting sequential association with membrane components and F-actin in the context of a fast overall turnover.

Orientation and Polarity in Collectively Migrating Cell Structures: Statics and Dynamics

Collective cell migration is often characterized by the spontaneous onset of multicellular protrusions (known as fingers) led by a single leader cell. Working with epithelial Madin-Darby canine kidney monolayers we show that cells within the fingers, as compared with the epithelium, are well oriented and polarized along the main finger direction, which suggests that these cells actively migrate. The cell orientation and polarity decrease continuously from the tip toward the epithelium over a penetration distance of typically two finger lengths. Furthermore, laser photoablation experiments at various locations along these fingers demonstrate that the cells in the fingers are submitted to a tensile stress whose value is larger close to the tip. From a dynamical point of view, cells entering a finger gradually polarize on timescales that depend upon their particular initial position. Selective laser nanosurgery of the leader lamellipodium shows not only that these structures need a leader to progress, but that this leader itself is the consequence of a prior self-organization of the cells forming the finger. These results highlight the complex interplay between the collective orientation within the fingers and the mechanical action of the leader.

Dynamic instability of the intracellular pressure drives bleb-based motility

We have demonstrated that the two- and three-dimensional motility of the human pathogenic parasite Entamoeba histolytica (Eh) depends on sustained instability of the intracellular hydrostatic pressure. This instability drives the cyclic generation and healing of membrane blebs, with typical protrusion velocities of 10–20 μm/second over a few hundred milliseconds and healing times of 10 seconds. The use of a novel micro-electroporation method to control the intracellular pressure enabled us to develop a qualitative model with three parameters: the rate of the myosin-driven internal pressure increase; the critical disjunction stress of membrane–cytoskeleton bonds; and the turnover time of the F-actin cortex. Although blebs occur randomly in space and irregularly time, they can be forced to occur with a defined periodicity in confined geometries, thus confirming our model. Given the highly efficient bleb-based motility of Eh in vitro and in vivo, Eh cells represent a unique model for studying the physical and biological aspects of amoeboid versus mesenchymal motility in two- and three-dimensional environments.

Myosin II and the Gal-GalNAc lectin play a crucial role in tissue invasion by Entamoeba histolytica

Entamoeba histolytica is the human parasite responsible of amoebiasis, during which highly motile trophozoites invade the intestinal epithelium leading to amoebic colitis, and disseminate via the blood circulation causing liver abscesses. The invasive process, central to the pathogenesis, is known to be driven by parasites motility. To investigate molecules responsible for in vivo motion, we performed a high resolution dynamic imaging analysis using two‐photon laser scanning microscopy. Image analysis of the parasites during invasion of Caco‐2 cell monolayers, an enterocyte‐like model, and hamster liver shows that E. histolytica undergoes non‐Brownian motion. However, studies of movements of parasite strains dominant negative for myosin II, a central component of the cytoskeleton, and for Gal‐GalNAc lectin, a major adhesion molecule, indicate that myosin II is essential for E. histolytica intercellular motility through intestinal cell monolayers and for its motility in liver. In contrast, the Gal‐GalNAc lectin exclusively triggers invasion of the liver. These observations are in agreement with emerging studies that highlight marked differences in the way that cells migrate in vitro in two dimensions versus in vivo in three dimensions. The approach that we have developed should be powerful to identify adhesive complexes required for in vivo cell migration in normal and pathogenic situations and may, thereby, lead to new therapeutic drug, for pathologies based on cell motility and adhesion.

Dynamical organization of the cytoskeletal cortex probed by micropipette aspiration

Bleb-based cell motility proceeds by the successive inflation and retraction of large spherical membrane protrusions (“blebs”) coupled with substrate adhesion. In addition to their role in motility, cellular blebs constitute a remarkable illustration of the dynamical interactions between the cytoskeletal cortex and the plasma membrane. Here we study the bleb-based motions of Entamoeba histolytica in the constrained geometry of a micropipette. We construct a generic theoretical model that combines the polymerization of an actin cortex underneath the plasma membrane with the myosin-generated contractile stress in the cortex and the stress-induced failure of membrane-cortex adhesion. One major parameter dictating the cell response to micropipette suction is the stationary cortex thickness, controlled by actin polymerization and depolymerization. The other relevant physical parameters can be combined into two characteristic cortex thicknesses for which the myosin stress (i) balances the suction pressure and (ii) provokes membrane-cortex unbinding. We propose a general phase diagram for cell motions inside a micropipette by comparing these three thicknesses. In particular, we theoretically predict and experimentally verify the existence of saltatory and oscillatory motions for a well-defined range of micropipette suction pressures.

Surface-Induced Polymerization of Actin

Living cells contain a very large amount of membrane surface area, which potentially influences the direction, the kinetics, and the localization of biochemical reactions. This paper quantitatively evaluates the possibility that a lipid monolayer can adsorb actin from a nonpolymerizing solution, induce its polymerization, and form a 2D network of individual actin filaments, in conditions that forbid bulk polymerization. G- and F-actin solutions were studied beneath saturated Langmuir monolayers containing phosphatidylcholine (PC, neutral) and stearylamine (SA, a positively charged surfactant) at PC:SA = 3:1 molar ratio. Ellipsometry, tensiometry, shear elastic measurements, electron microscopy, and dark-field light microscopy were used to characterize the adsorption kinetics and the interfacial polymerization of actin. In all cases studied, actin follows a monoexponential reaction-limited adsorption with similar time constants (approximately 10(3) s). At a longer time scale the shear elasticity of the monomeric actin adsorbate increases only in the presence of lipids, to a 2D shear elastic modulus of mu approximately 30 mN/m, indicating the formation of a structure coupled to the monolayer. Electron microscopy shows the formation of a 2D network of actin filaments at the PC:SA surface, and several arguments strongly suggest that this network is indeed causing the observed elasticity. Adsorption of F-actin to PC:SA leads more quickly to a slightly more rigid interface with a modulus of mu approximately 50 mN/m.

Crystallization of fluorescent quantum dots within a three-dimensional bio-organic template of actin filaments and lipid membranes.

Biological molecules and molecular self-assemblies are promising templates to organize well-defined inorganic nanostructures. We demonstrate the ability of a self-assembled three-dimensional crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots. Functionnalized tricystein peptidic quantum dots (QDs) are incorporated during the dynamical self-assembly of this actin/lipid template resulting in the formation of crystalline fibers. The crystal parameters, 26.5×18.9×35.5 nm3, are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. This process ensures the high quality of the crystal and results in unexpected fluorescence properties. This method of preparation offers opportunities to generate crystals with new symmetries and a large range of distance parameters.