Weak and transient lipid-protein interactions are thought to shape plasma membrane organization and function but have largely eluded experimental characterization. While model systems can only capture certain aspects of these interactions, extraction of unambiguous data from live cell experiments is challenging. We here ask a simple question directed at a fundamental aspect of plasma membrane organization: To what extent does a transmembrane protein influence, by its mere presence, the fluidity of its immediate lipid nanoenvironment? By specifically immobilizing proteins of interest at various densities in the live cell plasma membrane, we were able to determine its apparent in-plane hydrodynamic radius via quantification of the mobility reduction of individual lipid tracer molecules. In this assay, tight adhesion of lipid layers with reduced fluidity would manifest as an increased effective protein radius. We compared these values with structural biology data and used simulations to map the parameter space of possible nanoenvironment architectures around four different transmembrane proteins. For three of the four proteins tested, our data rule out the presence of tightly associated boundary lipids, calling into question their role as a general membrane-organizing principle.

Mechanical stimuli are an integral part of the natural cellular microenvironment, influencing cell growth, differentiation, and survival, particularly in mechanically challenging environments like tumors. These stimuli are also crucial in the T-cell microenvironment, where they play a role in antigen recognition and pathogen detection. To study T-cell mechanobiology effectively, in vitro methods must replicate these mechanical stimuli induced by compression, tension or shear flow, in the presence of antigen-presenting cells (APCs). While custom-made microdevices and microfluidic chips have successfully observed bulk cell behavior under mechanical strain, no existing device fully replicated the T-cell mechanoenvironment comprehensively. In this study, we developed a microdevice that integrates the mechanoenvironmental aspects of an APC mimicry with compression under live-cell imaging conditions. This device allows for precise confinement of cells between two glass surfaces, which can be individually coated with functional bio-interfaces. The microdevice is reusable and enables presetting of confinement heights, manual seeding of cells and the assembly of components directly at the microscope. To validate our microdevice we confined primary mouse T-cells on different APC-mimicking supported lipid bilayers while monitoring their morphology and migratory behaviour over time. To study the effect of confinement on TCR signalling, we tracked intracellular calcium levels and quantified Erk1/2 phosphorylation by immunostaining. We observed that T-cell morphology and motility are affected by confinement but also by bilayer composition. Moreover our findings suggest that confinement, despite not interfering with T-cell activation, might increase TCR background signalling in resting T-cells. Importantly, our microdevice is not limited to T-cell research; it can also serve as a platform for studying mechanical stimulation in other cell types, cell aggregates like spheroids and organoids, or even tissue samples in the presence of various bio-interfaces.

The interplay and communication between cells build the foundation of life. Many signaling processes at the cell surface and inside the cell, as well as the cellular function itself, depend on protein-protein interactions and the oligomerization of proteins. In the past, we presented an approach to single out interactions of fluorescently labeled membrane proteins by combining photobleaching and single-molecule microscopy. With this approach, termed "thinning out clusters while conserving the stoichiometry of labeling" (TOCCSL), oligomerization can be detected even at physiologically high surface densities of fluorescently labeled proteins. In TOCCSL, an aperture-restricted region of the plasma membrane is irreversibly photobleached by applying a high-intensity laser pulse. During a recovery time, in which illumination is turned off, nonphotobleached molecules from the nonilluminated area of the plasma membrane re-populate the aperture-restricted region. At the onset of this recovery process, these molecules can be detected as well-separated, diffraction-limited signals and their oligomerization state can be quantified. Here, we used extensive Monte Carlo simulations to provide a theoretical framework for quantitative interpretation of TOCCSL measurements. We determined the influence of experimental parameters and intrinsic characteristics of the investigated system on the outcome of a TOCCSL experiment. We identified the diffraction-affected laser intensity profile and the diffusion of molecules at the aperture edges during photobleaching as major sources of generating partially photobleached oligomers. They are falsely detected as lower-order oligomers and, hence, higher-order oligomers might be prevented from detection. The amount of partially photobleached oligomers that are analyzed depends on the photobleaching and the recovery time, on the mobility of molecules and—for mixed populations of oligomers—on mobility differences between different kinds of oligomers. Moreover, we quantified random colocalizations of molecules after recovery, which are falsely detected as higher-order oligomers.

Molecular crowding of agonist peptide/MHC class II complexes (pMHCIIs) with structurally similar, yet per se non-stimulatory endogenous pMHCIIs is postulated to sensitize T-cells for the recognition of single antigens on the surface of dendritic cells and B-cells. When testing this premise with the use of advanced live cell microscopy, we observe pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR engagement of highly abundant endogenous pMHCIIs is low or non-existent and affects neither TCR engagement of rare agonist pMHCII in early and advanced synapses nor agonist-induced TCR-proximal signaling. Our findings highlight the capacity of single freely diffusing agonist pMHCIIs to elicit the full T-cell response in an autonomous and peptide-specific fashion with consequences for adaptive immunity and immunotherapeutic approaches.

The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP 2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP 2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP 2 depletion; this indicates that PIP 2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP 2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP 2 directly regulates NET organization and function.

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.

T-cells are part of our adaptive immune system and are responsible for recognition of antigens in our bodies. T-cell activation is triggered upon binding of T-cell receptor (TCR) to major histocompatibility complex loaded with antigenic peptide (pMHC) which is presented on the surface of antigen presenting cells (APC). According to the kinetic segregation model of T-cell activation, T-cell topography plays a large role in the antigen recognition process. In this study we have applied a 3D superresolution method to study the spatial organization of the T-cell receptor within the immunological synapse with isotropic localization precision below 15 nm. The method combines stochastic optical reconstruction microscopy (STORM) with defocused imaging that exploits effects of the supercritical angle fluorescence on the shape of the point spread function. Additionally, we correlated the 3D superresolution images with diffraction limited images of the immune synapse obtained by interference reflection microscopy. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used our method to quantify membrane fluctuations and the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer. Our data show average distances of 18 nm up to 31 nm for activating and non-activating bilayers, respectively.

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. A variety of protein-protein interactions in trans-configuration defines the topography of the synapse and orchestrates the antigen-recognition process. In turn, the synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. For better understanding it is hence critical to map the 3D topography of the immunological synapse at high precision. Current methods, however, provide only rather coarse images of the protein distribution within the synapse, which do not reach the dimension of the protein ectodomains. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows 3-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and non-activating bilayers, respectively.

Determining nanoscale protein distribution via Photoactivation Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore's photophysics. If not accounted for, blinking of dyes on time-scales of typical PALM experiments will invariably cause overcounting artifacts, which become even more pronounced in fixed cells with predominantly immobile proteins. Here, we developed a lipid bilayer-based imaging platform as a means to determine the blinking behavior of two fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE of two photoswitchable 635). For all investigated fluorophores we revealed blinking cycles on time scales of several seconds. Our strategy is amenable to determining blinking signatures for any fluorophore of choice to support robust conclusions related to PALM. Furthermore, the gained blinking statistics can be utilized to discriminate clustered from randomly distributed membrane proteins.

Temperature has well-established immuno-modulating properties. Hypothermic temperatures are used in anti-inflammatory treatments, whereas hyperthermic temperatures can have immuno-stimulating effects (e.g. during fever). Yet, details on how temperature effects the immune system on a cellular level remain largely unrevealed. As a part of the adaptive immune system, T cells rapidly screen the surface of antigen presenting cells (APCs) for signs of infection or carcinogenesis. Together with signals from co-stimulatory proteins, the binding of the T cell receptor (TCR) to its cognate antigen (pMHC) on the APC surface triggers T cell activation and leads to the release of signalling molecules that direct the immune response. Here, we investigate the influence of temperature on T cell sensitivity during early T cell signalling. Supported lipid bilayers are used to mimic the APC surface in vitro. Ratiometric calcium imaging serves to identify activated T cells. This results in antigen dose-response curves that quantify T cell sensitivity. For varying thermal conditions, shifts in the dose-response relationship indicate temperature induced changes of T cell sensitivity. In addition, the experimental system allows for correlating T cell sensitivity with kinetic parameters of individual TCR-pMHC interactions.

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell-directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. Synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. It is hence important to determine the 3D topography of the immunological synapse at high precision. Current methods provide only rather coarse images of the protein distribution within the synapse. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows three-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and nonactivating bilayers, respectively.

T-cells are part of our adaptive immune system and are responsible for recognizing antigens in our body. T-cell activation is triggered by binding of the T-cell receptor (TCR) to antigenic peptide loaded major histocompatibility complexes (pMHC) presented on the surface of antigen presenting cells (APC), and the subsequent formation of the immunological synapse. According to the kinetic segregation model of T-cell activation, T-cell topography plays a significant role in this antigen recognition process. In our study we apply a 3D superresolution method to study the spatial organization of TCRs within the immunological synapse with an isotropic localization precision of 10 nm. The method combines stochastic optical reconstruction microscopy (STORM) with defocused imaging. The latter exploits the influence of the supercritical angle fluorescence on the shape of the point spread function. In order to mimic an APC surface, we functionalized a supported lipid bilayer with pMHC, co-stimulatory proteins and adhesion molecules. After addition of murine T-cells, the immunological synapse is imaged in total internal reflection fluorescence (TIRF) configuration. Our results reveal differences in the distance of the TCR to the supported lipid bilayer depending on the lateral position within the immunological synapse.

The stoichiometry of cell-surface proteins is fundamental to cellular signaling and function. Amongst others, particularly membrane proteins anchored to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) modification have been found to form dimeric or higher order oligomeric complexes. The role of this GPI-anchor in the oligomerization of membrane proteins and also the degree of clustering is highly discussed. Here, we present colocalization-based single-molecule microscopy studies to assess the oligomerization state of the GPI-anchored model protein SNAP-GPI. Since SNAP-tag proteins can bind only one Benzylguanine-linked dye, we achieve stoichiometric labeling of each GPI-anchored subunit by one out of two applied spectrally resolvable organic dye molecules. Dye concentrations were chosen such that approximately half of surface SNAP-GPI was labeled by one of each color. We utilized a 2-color version of "Thinning out clusters while conserving the stochiometry of labeling"(TOCCSL) to characterize the oligomerization state of SNAP-GPI at physiologically high surface densities in the live cell plasma membrane. In TOCCSL, a subregion of the cell-surface is stoichiometrically photobleached. At the onset of the recovery process, individual diffraction limited single-molecule signals can be imaged. In our co-localization study, we confirmed the presence of a high degree of SNAP-GPI dimerization, which was stable on the observable time scale of minutes. In agreement with our previous study which was based on brightness analysis, SNAP-GPI dimers vanished upon cholesterol depletion via the use of cholesteroloxidase. The use of TOCCSL also allowed for the application of single-molecule FRET microscopy, a technique based on non-radiative energy transfer between two spectrally different dyes which are in close proximity of each other. As FRET is extremely sensitive to small changes in distance it could be used to elucidate the spatial organization of SNAP-GPI dimers.

While single-molecule localization microscopy (SMLM) offers the invaluable prospect to visualize cellular structures below the diffraction limit of light microscopy, its potential has not yet been fully capitalized due to its inherent susceptibility to blinking artifacts. Particularly, overcounting of single molecule localizations has impeded a reliable and sensitive detection of biomolecular nanoclusters. Here we introduce a 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA), providing a parameter-free statistical framework for the qualitative analysis of two-dimensional SMLM data via significance testing methods. 2-CLASTA yields p-values for the null hypothesis of random biomolecular distributions, independent of the blinking behavior of the chosen fluorescent labels. The method is parameter-free and does not require any additional measurements nor grouping of localizations. We validated the method both by computer simulations as well as experimentally, using protein concatemers as a mimicry of biomolecular clustering. As the new approach is not affected by overcounting artifacts, it is able to detect biomolecular clustering of various shapes at high sensitivity down to a level of dimers.

Controlling the spatial organization of ligands on cell surfaces is emerging as a powerful tool to assess the relevance of multivalent binding, receptor clustering and molecular cooperativity for cell signaling. A mechanistic understanding of such processes would without doubt accelerate rational approaches in modern pharmacology and cell-based therapies.Here, we have designed a biomimetic interface based on lipid bilayer-anchored DNA origami nanostructures as a tool to study the spatial requirements for receptor-mediated signaling in cell-cell contacts. Additionally, our biointerface is responsive to dynamic receptor rearrangements on the cell surface upon ligand engagement as they typically occur in immunity, host-pathogen interactions as well as neuro-, developmental and cancer biology. Importantly, it allows the experimenter to adjust protein distances with nanoscale precision. We apply our approach to identify the role of nanoscale ligand arrangements for productive T-cell antigen receptor (TCR) signaling. While micron-sized clusters of TCRs and other signaling molecules have long been described to be associated with T-cell activation, recent research has implicated nanoclusters of a few TCRs as signaling-competent units. In the current study we were able to measure the molecular architecture of these signaling-competent units by taking advantage of the distinctive properties of our biointerface. Here, DNA origami function as nanoscale pegboards to position ligands, but act also as spacers to isolate individual ligand units by limiting their approach within clusters. We determine the minimal unit for T-cell triggering as two ligand-engaged TCRs spaced apart. This finding has wide-ranging implications for our mechanistic understanding regarding T-cell antigen recognition and, as a consequence, for the design of T-cell-based immunotherapies.