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.

Polarity in immune cells.

Immune cells are responsible for pathogen detection and elimination, as well as for signaling to other cells the presence of potential danger. In order to mount an efficient immune response, they need to move and search for a pathogen, interact with other cells, and diversify the population by asymmetric cell division. All these actions are regulated by cell polarity: cell polarity controls cell motility, which is crucial for scanning peripheral tissues to detect pathogens, and recruiting immune cells to sites of infection; immune cells, in particular lymphocytes, communicate with each other by a direct contact called immunological synapse, which entails a global polarization of the cell and plays a role in activating lymphocyte response; finally, immune cells divide asymmetrically from a precursor, generating a diversity of phenotypes and cell types among daughter cells, such as memory and effector cells.

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.

An Interesting Molecule: γ-Aminobutyric Acid. What Can We Learn from Hydra Polyps?

Neuronal excitability is controlled primarily by γ-aminobutyric acid (GABA) in the central and peripheral nervous systems of vertebrate as well as invertebrate organisms. Besides its recognized neurotransmitter functions, GABA also plays a fundamental role in neurogenesis and synaptogenesis during embryonic development. In addition, GABAergic mechanisms are also involved in disorders of various peripheral tissues, ranging from diabetes to hypothyroidism to inflammatory responses.

Traction Force Microscopy to Study B Lymphocyte Activation.

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor.

Diacylglycerol kinase ζ promotes actin cytoskeleton remodeling and mechanical forces at the B cell immune synapse.

Diacylglycerol kinases (DGKs) limit antigen receptor signaling in immune cells by consuming the second messenger diacylglycerol (DAG) to generate phosphatidic acid (PA). Here, we showed that DGKζ promotes lymphocyte function-associated antigen 1 (LFA-1)-mediated adhesion and F-actin generation at the immune synapse of B cells with antigen-presenting cells (APCs), mostly in a PA-dependent manner. Measurement of single-cell mechanical force generation indicated that DGKζ-deficient B cells exerted lower forces at the immune synapse than did wild-type B cells.

"If you please… draw me a cell". Insights from immune cells.

Studies in recent years have shed light on the particular features of cytoskeleton dynamics in immune cells, challenging the classical picture drawn from typical adherent cell lines. New mechanisms linking the dynamics of the membrane-cytoskeleton interface to the mechanical properties of immune cells have been uncovered and shown to be essential for immune surveillance functions. In this Essay, we discuss these features, and propose immune cells as a new playground for cell biologists who try to understand how cells adapt to different microenvironments to fulfil their functions efficiently.

Actomyosin-driven force patterning controls endocytosis at the immune synapse.

An important channel of cell-to-cell communication is direct contact. The immune synapse is a paradigmatic example of such type of interaction: it forms upon engagement of antigen receptors in lymphocytes by antigen-presenting cells and allows the local exchange of molecules and information. Although mechanics has been shown to play an important role in this process, how forces organize and impact on synapse function is unknown.

Immunocytochemical localization of a putative strychnine-sensitive glycine receptor in Hydra vulgaris.

Previous biochemical studies have identified strychnine-sensitive glycine receptors in membrane preparations of Hydra vulgaris (Cnidaria: Hydrozoa). Electrophysiological and behavioral evidence has shown that these receptors play a role in modulating pacemaker activity and feeding behavior. Here, we present our genomic analysis that revealed hydra proteins having strong homology with the strychnine-binding region of the human receptor protein, GlyRα1.

EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription.

Caveolae are small invaginated pits that function as dynamic mechanosensors to buffer tension variations at the plasma membrane. Here we show that under mechanical stress, the EHD2 ATPase is rapidly released from caveolae, SUMOylated, and translocated to the nucleus, where it regulates the transcription of several genes including those coding for caveolae constituents. We also found that EHD2 is required to maintain the caveolae reservoir at the plasma membrane during the variations of membrane tension induced by mechanical stress.

Immunochemical Localization of GABA Receptor Subunits in the Freshwater Polyp Hydra vulgaris (Cnidaria, Hydrozoa).

γ-aminobutyric acid (GABA) receptors, responding to GABA positive allosteric modulators, are present in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the most primitive metazoans to develop a nervous system. We examined the occurrence and distribution of GABA receptor subunits in Hydra tissues by western blot and immunohistochemistry. Antibodies against different GABA receptor subunits were used in Hydra membrane preparations.

Dynamics of the membrane-cytoskeleton interface in MHC class II-restricted antigen presentation.

Antigen presentation refers to the ability of cells to show MHC-associated determinants to T lymphocytes, leading to their activation. MHC class II molecules mainly present peptide-derived antigens that are internalized by endocytosis in antigen-presenting cells (APCs). Here, we describe how the interface between cellular membranes and the cytoskeleton regulates the various steps that lead to the presentation of exogenous antigens on MHC class II molecules in the two main types of APCs: dendritic cells (DCs) and B lymphocytes.

Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells.

Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined.

Regional modulation of the response to glutathione in Hydra vulgaris.

In the presence of prey, or upon exposure to reduced glutathione (GSH), Hydra polyps open a mouth to ingest the captured prey and close it after feeding; at rest the mouth is not evident. In previous papers we have shown that GABA, glycine and NMDA modulate the mechanisms of mouth closure through ligand-gated-ion-channel receptors that are similar to their mammalian analogues in terms of biochemical and pharmacological properties. In order to study the regional distribution of these receptors, we have applied the GSH assay to polyps amputated at different levels of the body column.

Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse.

B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR-antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse.

Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension.

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood.

How B cells capture, process and present antigens: a crucial role for cell polarity.

B cells are key components of the adaptive immune response. Their differentiation into either specific memory B cells or antibody-secreting plasma cells is a consequence of activation steps that involve the processing and presentation of antigens. The engagement of B cell receptors by surface-tethered antigens leads to the formation of an immunological synapse that coordinates cell signalling events and that promotes antigen uptake for presentation on MHC class II molecules.

Kinesin KIFC1 actively transports bare double-stranded DNA.

During the past years, exogenous DNA molecules have been used in gene and molecular therapy. At present, it is not known how these DNA molecules reach the cell nucleus. We used an in cell single-molecule approach to observe the motion of exogenous short DNA molecules in the cytoplasm of eukaryotic cells.

Coordinated modulation of cellular signaling through ligand-gated ion channels in Hydra vulgaris (Cnidaria, Hydrozoa).

Cnidarians lack well developed organs, but they have evolved the molecular and cellular components needed to assemble a nervous system. The apparent 'simplicity' of the cnidarian nervous net does not occur at the cellular level, but rather in the organisation of conducting systems. Cnidarian neurons are in fact electrically excitable, show the typical extended morphology and are connected by chemical synapses or gap junctions.

Polarized secretion of lysosomes at the B cell synapse couples antigen extraction to processing and presentation.

Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of a synapse that promotes Ag uptake for presentation onto major histocompatibility complex class II (MHCII) molecules. We have highlighted the membrane trafficking events and associated molecular mechanisms involved in Ag extraction and processing at the B cell synapse. MHCII-containing lysosomes are recruited to the synapse where they locally undergo exocytosis, allowing synapse acidification and the extracellular release of hydrolases that promote the extraction of the immobilized Ag.

Separation of time scales in one-dimensional directed nucleation-growth processes.

Proteins involved in homologous recombination such as RecA and hRad51 polymerize on single- and double-stranded DNA according to a nucleation-growth kinetics, which can be monitored by single-molecule in vitro assays. The basic models currently used to extract biochemical rates rely on ensemble averages and are typically based on an underlying process of bidirectional polymerization, in contrast with the often observed anisotropic polymerization of similar proteins. For these reasons, if one considers single-molecule experiments, the available models are useful to understand observations only in some regimes.