A prion-like domain is required for phase separation and chloroplast RNA processing during cold acclimation in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) plants can produce photosynthetic tissue with active chloroplasts at temperatures as low as 4°C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif domains of CP29A. The resulting droplets display liquid-like properties and are found near chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role in plant cold resistance.

SPI-1 virulence gene expression modulates motility of Salmonella Typhimurium in a proton motive force- and adhesins-dependent manner.

Both the bacterial flagellum and the evolutionary related injectisome encoded on the Salmonella pathogenicity island 1 (SPI-1) play crucial roles during the infection cycle of Salmonella species. The interplay of both is highlighted by the complex cross-regulation that includes transcriptional control of the flagellar master regulatory operon flhDC by HilD, the master regulator of SPI-1 gene expression. Contrary to the HilD-dependent activation of flagellar gene expression, we report here that activation of HilD resulted in a dramatic loss of motility, which was dependent on the presence of SPI-1. Single cell analyses revealed that HilD-activation triggers a SPI-1-dependent induction of the stringent response and a substantial decrease in proton motive force (PMF), while flagellation remains unaffected. We further found that HilD activation enhances the adhesion of Salmonella to epithelial cells. A transcriptome analysis revealed a simultaneous upregulation of several adhesin systems, which, when overproduced, phenocopied the HilD-induced motility defect. We propose a model where the SPI-1-dependent depletion of the PMF and the upregulation of adhesins upon HilD-activation enable flagellated Salmonella to rapidly modulate their motility during infection, thereby enabling efficient adhesion to host cells and delivery of effector proteins.

Maasi: A 3D printed spin coater with touchscreen.

Spin coaters are widely used to apply thin films of a material uniformly over a flat substrate. Despite the simplicity of this technique the entry price for such machines might be prohibitive, ranging from few hundreds to thousands of Euros. Here we present Maasi, an affordable alternative that is easy to build and has all functional key features to be used in a wide range of applications. Our design has a price of less than hundred Euros and an assembly time of only two hours. One of the key design principles was to use only 3D printed parts in combination with affordable Commercial Off-The-Shelf (COTS) components [1]. Reducing the complexity we use an electronic speed controller (ESC) with telemetry, to eliminate the need for a rotor position sensor [2]. A touchscreen further improves its usability, thus setting a perfect startpoint for the design of other affordable lab tools. The Maasi project includes different 3D printable substrate holders allowing treatment of formats up to 80 mm in diameter. We furthermore validate the Maasi spin coater by measuring its speed accuracy and performance for coating polydimethylsiloxane (PDMS) on glass coverslips for mechanobiological assays.

The solid tumor microenvironment-Breaking the barrier for T cells: How the solid tumor microenvironment influences T cells: How the solid tumor microenvironment influences T cells.

The tumor microenvironment (TME) plays a pivotal role in the behavior and development of solid tumors as well as shaping the immune response against them. As the tumor cells proliferate, the space they occupy and their physical interactions with the surrounding tissue increases. The growing tumor tissue becomes a complex dynamic structure, containing connective tissue, vascular structures, and extracellular matrix (ECM) that facilitates stimulation, oxygenation, and nutrition, necessary for its fast growth. Mechanical cues such as stiffness, solid stress, interstitial fluid pressure (IFP), matrix density, and microarchitecture influence cellular functions and ultimately tumor progression and metastasis. In this fight, our body is equipped with T cells as its spearhead against tumors. However, the altered biochemical and mechanical environment of the tumor niche affects T cell efficacy and leads to their exhaustion. Understanding the mechanobiological properties of the TME and their effects on T cells is key for developing novel adoptive tumor immunotherapies.

Understanding immune signaling using advanced imaging techniques.

Advanced imaging is key for visualizing the spatiotemporal regulation of immune signaling which is a complex process involving multiple players tightly regulated in space and time. Imaging techniques vary in their spatial resolution, spanning from nanometers to micrometers, and in their temporal resolution, ranging from microseconds to hours. In this review, we summarize state-of-the-art imaging methodologies and provide recent examples on how they helped to unravel the mysteries of immune signaling. Finally, we discuss the limitations of current technologies and share our insights on how to overcome these limitations to visualize immune signaling with unprecedented fidelity.

Nanoconfinement of microvilli alters gene expression and boosts T cell activation.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

Temporal analysis of T-cell receptor-imposed forces via quantitative single molecule FRET measurements.

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC, the physiological TCR-ligand. The sensor was tethered to planar glass-supported lipid bilayers (SLBs) and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid-phase SLBs instead, T-cells still exerted tensile forces yet of threefold reduced magnitude and only prior to but not upon activation.

Nuclear Leukocyte Immunoglobulin-like Receptor A3 Is Monomeric and Is Involved in Multiple Layers of Regulated Gene Expression and Translation.

The leukocyte immunoglobulin-like receptor A3 (LILRA3) is a soluble protein primarily expressed by peripheral blood monocytes and is abundant in sera of healthy donors. Extracellular LILRA3 is anti-inflammatory and displays neuro-regenerative functions in vitro. However, its intracellular expression, distribution, and function(s) remain unknown. Using a combination of high-resolution confocal and super-resolution microscopy, we identified intracellular expression of native LILRA3 in the nucleus of peripheral blood monocytes and in vitro-derived macrophages. This unexpected nuclear localization of LILRA3 was confirmed in LILRA3-GFP-transfected HEK293T cells. Western blot of proteins fractionated from primary macrophages and the transfected HEK293T cells confirmed nuclear localization of the native and expressed LILRA3 proteins. Interestingly, most of the LILRA3 in the nucleus was in a monomeric form like the biologically active secreted protein, while that in the other cellular compartments was in mixed monomeric, dimeric, and oligomeric forms. The predominant presence of monomeric LILRA3 in the nucleus was independently corroborated in transfected live HEK293T cells using the number and molecular brightness (N&B) analysis method. Immunoprecipitation and mass spectrometric peptide sequencing studies revealed that nuclear LILRA3 co-immunoprecipitated with several nuclear proteins involved in host protein synthesis machinery via direct interactions to a key multifunctional RNA-binding protein, the Ewing sarcoma breakpoint region 1 protein (EWS) (data are available via ProteomeXchange with identifier PXD024602). The biological significance of the nuclear expression of LILRA3 and its interaction with these key proteins remain to be elucidated.

NMR and EPR reveal a compaction of the RNA-binding protein FUS upon droplet formation.

Many RNA-binding proteins undergo liquid-liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)-a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients-can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron-electron resonance measurements revealed a compaction of FUS in the condensed phase.

External cues to drive B cell function towards immunotherapy.

Immunotherapy stands out as a powerful and promising therapeutic strategy in the treatment of cancer, infections, and autoimmune diseases. Adoptive immune therapies are usually centered on modified T cells and their specific expansion towards antigen-specific T cells against cancer and other diseases. However, despite their unmatched features, the potential of B cells in immunotherapy is just beginning to be explored. The main role of B cells in the immune response is to secrete antigen-specific antibodies and provide long-term protection against foreign pathogens. They further function as antigen-presenting cells (APCs) and secrete pro- and anti-inflammatory cytokines and thus exert positive and negative regulatory stimuli on other cells involved in the immune response such as T cells. Therefore, while hyperactivation of B cells can cause autoimmunity, their dysfunctions lead to severe immunodeficiencies. Only suitably activated B cells can play an active role in the treatment of cancers, infections, and autoimmune diseases. As a result, studies have focused on B cell-targeted immunotherapies in recent years. For this, the development, functions, interactions with the microenvironment, and clinical importance of B cells should be well understood. In this review, we summarize the main events during B cell activation. From the viewpoint of mechanobiology we discuss the translation of external cues such as surface topology, substrate stiffness, and biochemical signaling into B cell functions. We further dive into current B cell-targeted therapy strategies and their clinical applications. STATEMENT OF SIGNIFICANCE: B cells are proving as a promising tool in the field of immunotherapy. B cells exhibit various functions such as antibody production, antigen presentation or secretion of immune-regulatory factors which can be utilized in the fight against oncological or immunological disorders. In this review we discuss the importance of external mechanobiological cues such as surface topology, substrate stiffness, and biochemical signaling on B cell function. We further summarize B cell-targeted therapy strategies and their clinical applications, as in the context of anti-tumor responses and autoimmune diseases.

Functionalized Bead Assay to Measure Three-dimensional Traction Forces during T-cell Activation.

When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Here, we developed a traction force microscopy platform which allows for quantifying the pulls and pushes exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. We immobilized specific T-cell activating antibodies on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR, and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas CD45 is excluded from bead-microvilli contacts.

Localized detection of ions and biomolecules with a force-controlled scanning nanopore microscope.

Proteins, nucleic acids and ions secreted from single cells are the key signalling factors that determine the interaction of cells with their environment and the neighbouring cells. It is possible to study individual ion channels by pipette clamping, but it is difficult to dynamically monitor the activity of ion channels and transporters across the cellular membrane. Here we show that a solid-state nanopore integrated in an atomic force microscope can be used for the stochastic sensing of secreted molecules and the activity of ion channels in arbitrary locations both inside and outside a cell. The translocation of biomolecules and ions through the nanopore is observed in real time in live cells. The versatile nature of this approach allows us to detect specific biomolecules under controlled mechanical confinement and to monitor the ion-channel activities of single cells. Moreover, the nanopore microscope was used to image the surface of the nuclear membrane via high-resolution scanning ion conductance measurements.

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter.

The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by single-molecule microscopy that SERT forms stable higher-order oligomers of differing stoichiometry at the plasma membrane of living cells. Here, we report that SERT oligomer assembly at the endoplasmic reticulum (ER) membrane follows a dynamic equilibration process, characterized by rapid exchange of subunits between different oligomers, and by a concentration dependence of the degree of oligomerization. After trafficking to the plasma membrane, however, the SERT stoichiometry is fixed. Stabilization of the oligomeric SERT complexes is mediated by the direct binding to phosphoinositide phosphatidylinositol-4,5-biphosphate (PIP2). The observed spatial decoupling of oligomer formation from the site of oligomer operation provides cells with the ability to define protein quaternary structures independent of protein density at the cell surface.

Tuning membrane protein mobility by confinement into nanodomains.

High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.

Soluble LILRA3 promotes neurite outgrowth and synapses formation through a high-affinity interaction with Nogo 66.

Inhibitory proteins, particularly Nogo 66, a highly conserved 66-amino-acid loop of Nogo A (an isoform of RTN4), play key roles in limiting the intrinsic capacity of the central nervous system (CNS) to regenerate after injury. Ligation of surface Nogo receptors (NgRs) and/or leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue the paired immunoglobulin-like receptor B (PIRB) by Nogo 66 transduces inhibitory signals that potently inhibit neurite outgrowth. Here, we show that soluble leukocyte immunoglobulin-like receptor A3 (LILRA3) is a high-affinity receptor for Nogo 66, suggesting that LILRA3 might be a competitive antagonist to these cell surface inhibitory receptors. Consistent with this, LILRA3 significantly reversed Nogo-66-mediated inhibition of neurite outgrowth and promoted synapse formation in primary cortical neurons through regulation of the ERK/MEK pathway. LILRA3 represents a new antagonist to Nogo-66-mediated inhibition of neurite outgrowth in the CNS, a function distinct from its immune-regulatory role in leukocytes. This report is also the first to demonstrate that a member of LILR family normally not expressed in rodents exerts functions on mouse neurons through the highly homologous Nogo 66 ligand.

Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix.

Despite the crucial role of extracellular matrix (ECM) in directing cell fate in healthy and diseased tissues--particularly in development, wound healing, tissue regeneration and cancer--the mechanisms that direct the assembly and regulate hierarchical architectures of ECM are poorly understood. Collagen I matrix assembly in vivo requires active fibronectin (Fn) fibrillogenesis by cells. Here we exploit Fn-FRET probes as mechanical strain sensors and demonstrate that collagen I fibres preferentially co-localize with more-relaxed Fn fibrils in the ECM of fibroblasts in cell culture. Fibre stretch-assay studies reveal that collagen I's Fn-binding domain is responsible for the mechano-regulated interaction. Furthermore, we show that Fn-collagen interactions are reciprocal: relaxed Fn fibrils act as multivalent templates for collagen assembly, but once assembled, collagen fibres shield Fn fibres from being stretched by cellular traction forces. Thus, in addition to the well-recognized, force-regulated, cell-matrix interactions, forces also tune the interactions between different structural ECM components.

Superresolution microscopy reveals spatial separation of UCP4 and F0F1-ATP synthase in neuronal mitochondria.

Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regulation of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical reconstruction microscopy) to visualize several mitochondrial proteins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F0F1-ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F0F1-ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various expression levels in different mitochondria, supporting the hypothesis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F0F1-ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from acting as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production.

The ultrastructure of fibronectin fibers pulled from a protein monolayer at the air-liquid interface and the mechanism of the sheet-to-fiber transition.

Fibronectin is a globular protein that circulates in the blood and undergoes fibrillogenesis if stretched or under other partially denaturing conditions, even in the absence of cells. Stretch assays made by pulling fibers from droplets of solutions containing high concentrations of fibronectin have previously been introduced in mechanobiology, particularly to ask how bacteria and cells exploit the stretching of fibronectin fibers within extracellular matrix to mechano-regulate its chemical display. Our electron microscopy analysis of their ultrastructure now reveals that the manually pulled fibronectin fibers are composed of densely packed lamellar spirals, whose interlamellar distances are dictated by ion-tunable electrostatic interactions. Our findings suggest that fibrillogenesis proceeds via an irreversible sheet-to-fiber transition as the fibronectin sheet formed at the air-liquid interface of the droplet is pulled off by a sharp tip. This far from equilibrium process is driven by the externally applied force, interfacial surface tension, shear-induced fibronectin self-association, and capillary force-induced buffer drainage. The ultrastructural characterization is then contrasted with previous FRET studies that characterized the molecular strain within these manually pulled fibers. Particularly relevant for stretch-dependent binding studies is the finding that the interior fiber surfaces are accessible to nanoparticles smaller than 10 nm. In summary, our study discovers the underpinning mechanism by which highly hierarchically structured fibers can be generated with unique mechanical and mechano-chemical properties, a concept that might be extended to other bio- or biomimetic polymers.

Do mechanical forces contribute to nanoscale membrane organisation in T cells?

Mechanotransduction describes how a cell senses and interacts with its environment. The concept originated in adhesion biology where adhesion receptors, integrins, facilitate force transmission between the extracellular matrix and the intracellular actin cytoskeleton. Indeed, during any adhesive contacts, cells do exert mechanical force. Hence, the probing of the local environment by cells results in mechanical cues that contribute to cellular functions and cell fate decisions such as migration, proliferation, differentiation and apoptosis. On the molecular level, mechanical forces can rearrange proteins laterally within the membrane, regulate their activity by inducing conformational changes and probe the mechanical properties and bond strength of receptor-ligands. From this point of view, it appears surprising that molecular forces have been largely overlooked in membrane organisation and ligand discrimination processes in lymphocytes. During T cell activation, the T cell receptor recognises and distinguishes antigenic from benign endogenous peptides to initiate the reorganisation of membrane proteins into signalling clusters within the immunological synapse. In this review, we asked whether characteristics of fibroblast force sensing could be applied to immune cell antigen recognition and signalling, and outline state-of-the-art experimental strategies for studying forces in the context of membrane organisation. This article is part of a Special Issue entitled: Nanoscale membrane orgainisation and signalling.