A simple method to make, trap and deform a vesicle in a gel.

We present a simple method to produce giant lipid pseudo-vesicles (vesicles with an oily cap on the top), trapped in an agarose gel. The method can be implemented using only a regular micropipette and relies on the formation of a water/oil/water double droplet in liquid agarose. We characterize the produced vesicle with fluorescence imaging and establish the presence and integrity of the lipid bilayer by the successful insertion of [Formula: see text]-Hemolysin transmembrane proteins. Finally, we show that the vesicle can be easily mechanically deformed, non-intrusively, by indenting the surface of the gel.

Adhesion as a trigger of droplet polarization in flowing emulsions.

Tissues are subjected to large external forces and undergo global deformations during morphogenesis. We use synthetic analogues of tissues to study the impact of cell-cell adhesion on the response of cohesive cellular assemblies under such stresses. In particular, we use biomimetic emulsions in which the droplets are functionalized in order to exhibit specific droplet-droplet adhesion. We flow these emulsions in microfluidic constrictions and study their response to this forced deformation via confocal microscopy. We find that the distributions of avalanche sizes are conserved between repulsive and adhesive droplets. However, adhesion locally impairs the rupture of droplet-droplet contacts, which in turn pulls on the rearranging droplets. As a result, adhesive droplets are a lot more deformed along the axis of elongation in the constriction. This finding could shed light on the origin of polarization processes during morphogenesis.

Depletion attraction impairs the plasticity of emulsions flowing in a constriction.

We study the elasto-plastic behavior of dense attractive emulsions under a mechanical perturbation. The attraction is introduced through non-specific depletion interactions between the droplets and is controlled by changing the concentration of surfactant micelles in the continuous phase. We find that such attractive forces are not sufficient to induce any measurable modification on the scalings between the local packing fraction and the deformation of the droplets. However, when the emulsions are flowed through 2D microfluidic constrictions, we uncover a measurable effect of attraction on their elasto-plastic response. Indeed, we measure higher levels of deformation inside the constriction for attractive droplets. In addition, we show that these measurements correlate with droplet rearrangements that are spatially delayed in the constriction for higher attraction forces.

A Multiparametric and High-Throughput Assay to Quantify the Influence of Target Size on Phagocytosis.

Phagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. It consists in the uptake of pathogenic or cellular targets larger than 0.5 µm. For the biggest particles, the phagocytic process involves a massive reorganization of membrane and actin cytoskeleton as well as an important intracellular deformation all in a matter of minutes. The study of the role of the size of objects in their phagocytosis has led to contradictory results in the last decades. We designed a method using confocal microscopy, automated image analysis, and databases for fast quantitative analysis of phagocytosis assays. It yields comprehensive data on the cells and targets geometric and fluorescence intensity parameters, automatically discriminates internalized from external targets, and stores the relationship between a cell and the targets it has engulfed. We used two types of targets (solid polystyrene beads and liquid lipid droplets) to investigate the influence of size on the phagocytic uptake of macrophages. The method made it possible not only to perform phagocytic assays with functionalized droplets and beads of different sizes but to use polydisperse particles to further our understanding of the role of size in phagocytosis. The use of monodisperse and polydisperse objects shows that whereas smaller monodisperse objects are internalized in greater numbers, objects of different sizes presented simultaneously are internalized without preferred size. The total surface engulfed by the cell is thus the main factor limiting the uptake of particles, regardless of their nature or size. A meta-analysis of the literature reveals that this dependence in surface is consistently conserved throughout cell types, targets' nature, or activated receptors.

The nature and intensity of mechanical stimulation drive different dynamics of MRTF-A nuclear redistribution after actin remodeling in myoblasts.

Serum response factor and its cofactor myocardin-related transcription factor (MRTF) are key elements of muscle-mass adaptation to workload. The transcription of target genes is activated when MRTF is present in the nucleus. The localization of MRTF is controlled by its binding to G-actin. Thus, the pathway can be mechanically activated through the mechanosensitivity of the actin cytoskeleton. The pathway has been widely investigated from a biochemical point of view, but its mechanical activation and the timescales involved are poorly understood. Here, we applied local and global mechanical cues to myoblasts through two custom-built set-ups, magnetic tweezers and stretchable substrates. Both induced nuclear accumulation of MRTF-A. However, the dynamics of the response varied with the nature and level of mechanical stimulation and correlated with the polymerization of different actin sub-structures. Local repeated force induced local actin polymerization and nuclear accumulation of MRTF-A by 30 minutes, whereas a global static strain induced both rapid (minutes) transient nuclear accumulation, associated with the polymerization of an actin cap above the nucleus, and long-term accumulation, with a global increase in polymerized actin. Conversely, high strain induced actin depolymerization at intermediate times, associated with cytoplasmic MRTF accumulation.

Mannose-Coated Fluorescent Lipid Microparticles for Specific Cellular Targeting and Internalization via Glycoreceptor-Induced Phagocytosis.

In this work, we report on the development of mannose-coated fluorescent lipid microparticles to study the role of C-type lectin membrane receptors in phagocytosis. The micrometric droplets of soybean oil-in-water emulsion were functionalized with a tailor-made fluorescent mannolipid. The amphiphilic ligand was built from a mannose unit, a lipid C11 spacer, and a naphthalimide fluorophore. The functionalization of the droplets was monitored by fluorescence microscopy as well as their interaction with concanavalin A, which was used as a model lectin in vitro. The use of a monovalent ligand on the surface of emulsion droplets yielded particles with an affinity approximately 40 times higher than that of free mannose. In cellulo, the coated droplets were shown to be specifically internalized by macrophages in a receptor-dependent phagocytic pathway. The naked droplets, on the other hand, displayed very little internalization because of their low immunogenicity. This work thus brings evidence that C-type lectin membrane receptors may act as phagocytic receptors. The functionalization of the droplets with the tailored amphiphilic fluorescent ligand also provides insights into the development of organic fluorescent particles that may prove useful for developing targeted imaging and delivery tools.

The desmin network is a determinant of the cytoplasmic stiffness of myoblasts.

BACKGROUND INFORMATION: The mechanical properties of cells are essential to maintain their proper functions, and mainly rely on their cytoskeleton. A lot of attention has been paid to actin filaments, demonstrating their central role in the cells mechanical properties, but much less is known about the participation of intermediate filament (IF) networks. Indeed the contribution of IFs, such as vimentin, keratins and lamins, to cell mechanics has only been assessed recently. We study here the involvement of desmin, an IF specifically expressed in muscle cells, in the rheology of immature muscle cells. Desmin can carry mutations responsible for a class of muscle pathologies named desminopathies. RESULTS: In this study, using three types of cell rheometers, we assess the consequences of expressing wild-type (WT) or mutated desmin on the rheological properties of single myoblasts. We find that the mechanical properties of the cell cortex are not correlated to the quantity, nor the quality of desmin expressed. On the contrary, the overall cell stiffness increases when the amount of WT or mutated desmin polymerised in cytoplasmic networks increases. However, myoblasts become softer when the desmin network is partially depleted by the formation of aggregates induced by the expression of a desmin mutant. CONCLUSIONS: We demonstrate that desmin plays a negligible role in the mechanical properties of the cell cortex but is a determinant of the overall cell stiffness. More particularly, desmin participates to the cytoplasm viscoelasticity. SIGNIFICANCE: Desminopathies are associated with muscular weaknesses attributed to a disorganisation of the structure of striated muscle that impairs the active force generation. The present study evidences for the first time the key role of desmin in the rheological properties of myoblasts, raising the hypothesis that desmin mutations could also alter the passive mechanical properties of muscles, thus participating to the lack of force build up in muscle tissue.

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell-cell contact fluidity.

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell-cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell-cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell-cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell-cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell-cell contact fluidity.

Plate coating: influence of concentrated surfactants on the film thickness.

We present a large range of experimental data concerning the influence of surfactants on the well-known Landau-Levich-Derjaguin experiment where a liquid film is generated by pulling a plate out of a bath. The thickness h of the film was measured as a function of the pulling velocity V for different kinds of surfactants (C(12)E(6), which is a nonionic surfactant, and DeTAB and DTAB, which are ionic) and at various concentrations near and above the critical micellar concentration (cmc). We report the thickening factor α = h/h(LLD), where h(LLD) is the film thickness obtained without a surfactant effect, i.e., as for a pure fluid but with the same viscosity and surface tension as the surfactant solution, over a wide range of capillary numbers (Ca = ηV/γ, with η being the surfactant solution viscosity and γ its surface tension) and identify three regimes: (i) at small Ca α is large due to confinement and surface elasticity (or Marangoni) effects, (ii) for increasing Ca there is an intermediate regime where α decreases as Ca increases, and (iii) at larger (but still small) Ca α is slightly higher than unity due to surface viscosity effects. In the case of nonionic surfactants, the second regime begins at a fixed Ca, independent of the surfactant concentration, while for ionic surfactants the transition depends on the concentration, which we suggest is probably due to the existence of an electrostatic barrier to surface adsorption. Control of the physical chemistry at the interface allowed us to elucidate the nature of the three regimes in terms of surface rheological properties.