FRET-Sensing of Multivalent Protein Binding at the Interface of Biomimetic Microparticles Functionalized with Fluorescent Glycolipids.

Cell adhesion is a central process in cellular communication and regulation. Adhesion sites are triggered by specific ligand-receptor interactions inducing the clustering of both partners at the contact point. Investigating cell adhesion using microscopy techniques requires targeted fluorescent particles with a signal sensitive to the clustering of receptors and ligands at the interface. Herein, we report on simple cell or bacterial mimics, based on liquid microparticles made of lipiodol functionalized with custom-designed fluorescent lipids. These lipids are targeted toward lectins or biotin membrane receptors, and the resulting particles can be specifically identified and internalized by cells, as demonstrated by their phagocytosis in primary murine bone marrow-derived macrophages. We also evidence the possibility to sense the binding of a multivalent lectin, concanavalin A, in solution by monitoring the energy transfer between two matching fluorescent lipids on the surface of the particles. We anticipate that these liquid particle-based sensors, which are able to report via Förster resonance energy transfer (FRET) on the movement of ligands on their interface upon protein binding, will provide a useful tool to study receptor binding and cooperation during adhesion processes such as phagocytosis.

Functionalized Lipid Droplets and Microfluidics Approach to Study Immune Cell Polarity In Vitro.

The study of lymphocyte polarization upon antigen encounter typically relies on the random pairing between the cells of interest and a stimulating particle (micro bead) that mimics only some of the properties of the antigen-presenting cells. Here, we show how to build and use a microfluidic chip that allows to multiplex and synchronize the encounter between a lymphocyte and an antigen-presenting object: a functionalized oil-in-water droplet. We also explain how to fabricate and functionalize lipid droplets, an antigen-presenting tool that is, at the same time, deformable, fluid, and spherical.

Phenotyping polarization dynamics of immune cells using a lipid droplet-cell pairing microfluidic platform.

The immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intracellular components necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied. Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how ligand concentration, surface fluidity, and substrate rigidity impact lysosome polarization, we show that its onset and kinetics depend on the local antigen concentration at the synapse and on substrate rigidity. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout.

Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes.

Immune synapse formation is a key step for lymphocyte activation. In B lymphocytes, the immune synapse controls the production of high-affinity antibodies, thereby defining the efficiency of humoral immune responses. While the key roles played by both the actin and microtubule cytoskeletons in the formation and function of the immune synapse have become increasingly clear, how the different events involved in synapse formation are coordinated in space and time by actin-microtubule interactions is not understood. Using a microfluidic pairing device, we studied with unprecedented resolution the dynamics of the various events leading to immune synapse formation and maintenance in murine B cells. Our results identify two groups of events, local and global, dominated by actin and microtubules dynamics, respectively. They further highlight an unexpected role for microtubules and the GEF-H1-RhoA axis in restricting F-actin polymerization at the lymphocyte-antigen contact site, thereby allowing the formation and maintenance of a unique competent immune synapse.

Design of a comprehensive microfluidic and microscopic toolbox for the ultra-wide spatio-temporal study of plant protoplasts development and physiology.

BACKGROUND: Plant protoplasts are basic plant cells units in which the pecto-cellulosic cell wall has been removed, but the plasma membrane is intact. One of the main features of plant cells is their strong plasticity, and their propensity to regenerate an organism from a single cell. Methods and differentiation protocols used in plant physiology and biology usually involve macroscopic vessels and containers that make difficult, for example, to follow the fate of the same protoplast all along its full development cycle, but also to perform continuous studies of the influence of various gradients in this context. These limits have hampered the precise study of regeneration processes. RESULTS: Herein, we present the design of a comprehensive, physiologically relevant, easy-to-use and low-cost microfluidic and microscopic setup for the monitoring of Physcomitrella patens (P. patens) growth and development on a long-term basis. The experimental solution we developed is made of two parts (i) a microfluidic chip composed of a single layer of about a hundred flow-through microfluidic traps for the immobilization of protoplasts, and (ii) a low-cost, light-controlled, custom-made microscope allowing the continuous recording of the moss development in physiological conditions. We validated the experimental setup with three proofs of concepts: (i) the kinetic monitoring of first division steps and cell wall regeneration, (ii) the influence of the photoperiod on growth of the protonemata, and (iii) finally the induction of leafy buds using a phytohormone, cytokinin. CONCLUSIONS: We developed the design of a comprehensive, physiologically relevant, easy-to-use and low-cost experimental setup for the study of P. patens development in a microfluidic environment. This setup allows imaging of P. patens development at high resolution and over long time periods.

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.

Point Mutations Lead to Increased Levels of c-di-GMP and Phenotypic Changes to the Colony Biofilm Morphology in Alcanivorax borkumensis SK2.

Alcanivorax borkumensis is a ubiquitous marine bacterium that utilizes alkanes as a sole carbon source. We observed two phenotypes in the A. borkumensis SK2 type strain: rough (R) and smooth (S) types. The S type exhibited lower motility and higher polysaccharide production than the R type. Full genome sequencing revealed a mutation in the S type involved in cyclic-di-GMP production. The present results suggest that higher c-di-GMP levels in the S type control the biofilm forming behavior of this bacterium in a manner commensurate with other Gram-negative bacteria.

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.

Nanometric emulsions encapsulating solid particles as alternative carriers for intracellular delivery.

AIM: Formulate nanometric oil droplets for encapsulating solid nanoparticles and assess their interactions with cells. MATERIALS & METHODS: Soybean oil droplets, stabilized by Pluronic F68 surfactant, incorporating hydrophobically modified fluorescent silica, nanoparticles were obtained. Cytotoxicity over time, internalization, subsequent intracellular localization and internalization pathways were assessed by microscopy (fluoresence and TEM) in vitro with HeLa cells. RESULTS: Oil droplets encapsulating solid nanoparticles are readily internalized by HeLa cells like free nanoparticles but the intracellular localization differs (nanoemulsions less colocalized with lysosomes) as well as internalization pathway is used (nanoemulsions partially internalized by nonendocytic transport). No cytotoxicity could be observed for either particles tested. CONCLUSION: Our results confirm that nanometric emulsions encapsulating solid nanoparticles can be used for alternative and multifunctional intracellular delivery.

Phagocytosis of immunoglobulin-coated emulsion droplets.

Phagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. The size of targets to be eliminated ranges from small particles as bacteria to large objects as cancerous or senescent cells. Most of our current quantitative knowledge on phagocytosis is based on the use of solid polymer microparticles as model targets that are well adapted to the study of phagocytosis mechanisms that do not involve any lateral mobility of the ligands, despite the relevance of this parameter in the immunological context. Herein we designed monodisperse, IgG-coated emulsion droplets that are efficiently and specifically internalized by macrophages through in-vitro FcγR-mediated phagocytosis. We show that, contrary to solid polymeric beads, droplet uptake is efficient even for low IgG densities, and is accompagnied by the clustering of the opsonins in the zone of contact with the macrophage during the adhesion step. Beyond the sole interest in the design of the material, our results suggest that lateral mobility of proteins at the interface of a target greatly enhances the phagocytic uptake.

Active transport of oil droplets along oriented microtubules by kinesin molecular motors.

We demonstrate the active transport of liquid cargos in the form of oil-in-water emulsion droplets loaded on kinesin motor proteins moving along oriented microtubules. We analyze the motility properties of the kinesin motors (velocity and run length) and find that the liquid cargo in the form of oil droplets does not alter the motor function of the kinesin molecules. This work provides a novel method for handling only a few molecules/particles encapsulated inside the oil droplets and represents a key finding for the integration of kinesin-based active transport into nanoscale lab-on-a-chip devices. We also investigate the effect of the diameter of the droplets on the motility properties of the kinesin motors. The velocity is approximately constant irrespective of the diameter of the droplets whereas we highlight a strong increase of the run length when the diameter of the droplets increases. We correlate these results with the number of kinesin motors involved in the transport process and find an excellent agreement between our experimental result and a theoretical model.

Encapsulation of magnetic and fluorescent nanoparticles in emulsion droplets.

Oils containing both fluorescent semiconductor and magnetic oxide nanoparticles are used to produce oil in water emulsions. This technique produces oil droplets with homogeneous fluorescence and high magnetic nanoparticle concentrations. The optical properties of the oil droplets are studied as a function of the droplet sizes for various concentrations of fluorescent and magnetic nanoparticles. For all concentrations tested, we find a linear variation of the droplet fluorescent intensity as a function of the droplet volume. For a given size and a given quantum dot (QD) concentration, the droplet fluorescence intensity drops sharply as a function of the magnetic nanoparticle concentration. We show that this decrease is due mainly to the strong absorption cross section of the magnetic nanoparticles and to a lesser extent to the dynamic and static quenching of the QD fluorescence. The role of the iron oxide nanoparticle localization in the droplet (surface versus volume) is also discussed.