TCRs are randomly distributed on the plasma membrane of resting antigen-experienced T cells.

The main function of T cells is to identify harmful antigens as quickly and precisely as possible. Super-resolution microscopy data have indicated that global clustering of T cell antigen receptors (TCRs) occurs before T cell activation. Such pre-activation clustering has been interpreted as representing a potential regulatory mechanism that fine tunes the T cell response. We found here that apparent TCR nanoclustering could be attributed to overcounting artifacts inherent to single-molecule-localization microscopy. Using complementary super-resolution approaches and statistical image analysis, we found no indication of global nanoclustering of TCRs on antigen-experienced CD4+ T cells under non-activating conditions. We also used extensive simulations of super-resolution images to provide quantitative limits for the degree of randomness of the TCR distribution. Together our results suggest that the distribution of TCRs on the plasma membrane is optimized for fast recognition of antigen in the first phase of T cell activation.

Monomeric TCRs drive T cell antigen recognition.

T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements. We found exclusively monomeric TCR-CD3 complexes driving the recognition of antigenic pMHCs, which underscores the exceptional capacity of single TCR-CD3 complexes to elicit robust intracellular signaling.

T-cell antigen recognition: catch-as-catch-can or catch-22?

T-cell antigen recognition is invariably affected by tensile forces as they are exerted on T-cell antigen receptors (TCRs) transiently binding antigenic peptide/MHC complexes. In this issue of The EMBO Journal, Pettmann and colleagues promote the concept that forces reduce the lifetime of more stable stimulatory TCR-pMHC interactions to a larger extent than that of less stable non-stimulatory TCR-pMHC interactions. The authors argue that forces impede rather than boost T-cell antigen discrimination, which is promoted by force-shielding within the immunological synapse through cell adhesion via CD2/CD58 and LFA-1/ICAM-1.

Monomeric agonist peptide/MHCII complexes activate T-cells in an autonomous fashion.

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 asymmetric plasma membrane-A composite material combining different functionalities?: Balancing Barrier Function and Fluidity for Effective Signaling.

One persistent puzzle in the life sciences is the asymmetric lipid composition of the cellular plasma membrane: while the exoplasmic leaflet is enriched in lipids carrying predominantly saturated fatty acids, the cytoplasmic leaflet hosts preferentially lipids with (poly-)unsaturated fatty acids. Given the high energy requirements necessary for cells to maintain this asymmetry, the question naturally arises regarding its inherent benefits. In this paper, we propose asymmetry to represent a potential solution for harmonizing two conflicting requirements for the plasma membrane: first, the need to build a barrier for the uncontrolled influx or efflux of substances; and second, the need to form a fluid and dynamic two-dimensional substrate for signaling processes. We hence view here the plasma membrane as a composite material, where the exoplasmic leaflet is mainly responsible for the functional integrity of the barrier and the cytoplasmic leaflet for fluidity. We reinforce the validity of the proposed mechanism by presenting quantitative data from the literature, along with multiple examples that bolster our model.

Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants.

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/mitogen-activated protein kinase pathway by their tyrosine kinase activation known to determine cell growth, tissue differentiation, and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here, we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (monomeric GFP [mGFP]-FGFR3) in certain areas of the plasma membrane of living cells. We quantified receptor activation via total internal reflection fluorescence microscopy of FGFR3 signaling at the cell membrane that captured the recruitment of the downstream signal transducer growth factor receptor-bound 2 (GRB2) tagged with mScarlet (GRB2-mScarlet) to FGFR3 micropatterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) for WT and four FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, and K650E). Our data showed that ligand addition increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared with previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This can be attributed to the different methodology, since micropatterning specifically captures signaling events at the plasma membrane. Collectively, our results provide further insight into the functional effects of mutations to FGFR3.

DNA origami demonstrate the unique stimulatory power of single pMHCs as T cell antigens.

T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. When targeting TCRs via stably binding monovalent antibody fragments, we found the minimum signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC molecules.

Cathelicidin and PMB neutralize endotoxins by multifactorial mechanisms including LPS interaction and targeting of host cell membranes.

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.

Monte Carlo simulations for the evaluation of oligomerization data in TOCCSL experiments.

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.

Three-Dimensional Single Molecule Localization Microscopy Reveals the Topography of the Immunological Synapse at Isotropic Precision below 15 nm.

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.

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.

SLC6 transporter oligomerization.

Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix-spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre-fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release-type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions.

Single-molecule optical absorption imaging by nanomechanical photothermal sensing.

Absorption microscopy is a promising alternative to fluorescence microscopy for single-molecule imaging. So far, molecular absorption has been probed optically via the attenuation of a probing laser or via photothermal effects. The sensitivity of optical probing is not only restricted by background scattering but it is fundamentally limited by laser shot noise, which minimizes the achievable single-molecule signal-to-noise ratio. Here, we present nanomechanical photothermal microscopy, which overcomes the scattering and shot-noise limit by detecting the photothermal heating of the sample directly with a temperature-sensitive substrate. We use nanomechanical silicon nitride drums, whose resonant frequency detunes with local heating. Individual Au nanoparticles with diameters from 10 to 200 nm and single molecules (Atto 633) are scanned with a heating laser with a peak irradiance of 354 ± 45 µW/µm2 using 50× long-working-distance objective. With a stress-optimized drum we reach a sensitivity of 16 fW/Hz1/2 at room temperature, resulting in a single-molecule signal-to-noise ratio of >70. The high sensitivity combined with the inherent wavelength independence of the nanomechanical sensor presents a competitive alternative to established tools for the analysis and localization of nonfluorescent single molecules and nanoparticles.

Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate-independent manner.

The human dopamine transporter (hDAT) regulates the level of the neurotransmitter dopamine (DA) in the synaptic cleft and recycles DA for storage in the presynaptic vesicular pool. Many neurotransmitter transporters exist as oligomers, but the physiological role of oligomerization remains unclear; for example, it has been speculated to be a prerequisite for amphetamine-induced release and protein trafficking. Previous studies point to an oligomeric quaternary structure of hDAT; however, the exact stoichiometry and the fraction of co-existing oligomeric states are not known. Here, we used single-molecule brightness analysis to quantify the degree of oligomerization of heterologously expressed hDAT fused to monomeric GFP (mGFP-hDAT) in Chinese hamster ovary (CHO) cells. We observed that monomers and dimers of mGFP-hDAT co-exist and that higher-order molecular complexes of mGFP-hDAT are absent at the plasma membrane. The mGFP-hDAT dimers were stable over several minutes, and the fraction of dimers was independent of the mGFP-hDAT surface density. Furthermore, neither oxidation nor depletion of cholesterol had any effect on the fraction of dimers. Unlike for the human serotonin transporter (hSERT), in which direct binding of phosphatidylinositol 4,5-bisphosphate (PIP2) stabilized the oligomers, the stability of mGFP-hDAT dimers was PIP2 independent.

Single-Molecule Localization Microscopy to Study Protein Organization in the Filamentous Fungus Trichoderma atroviride.

Single-molecule localization microscopy has boosted our understanding of biological samples by offering access to subdiffraction resolution using fluorescence microscopy methods. While in standard mammalian cells this approach has found wide-spread use, its application to filamentous fungi has been scarce. This is mainly due to experimental challenges that lead to high amounts of background signal because of ample autofluorescence. Here, we report the optimization of labeling, imaging and data analysis protocols to yield the first single-molecule localization microscopy images of the filamentous fungus Trichoderma atroviride. As an example, we show the spatial distribution of the Sur7 tetraspanin-family protein Sfp2 required for hyphal growth and cell wall stability in this mycoparasitic fungus.

Homo- and Heteroassociations Drive Activation of ErbB3.

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 co-expression, a small fraction was present as constitutive homodimers exhibiting a ∼40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced the mobility of homodimers fourfold compared to constitutive homodimers. Expression of ErbB2 elevated the homodimeric fraction of ErbB3 even in unstimulated cells and induced a ∼2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization arm of ErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling.

Varying label density allows artifact-free analysis of membrane-protein nanoclusters.

We present a method to robustly discriminate clustered from randomly distributed molecules detected with techniques based on single-molecule localization microscopy, such as PALM and STORM. The approach is based on deliberate variation of labeling density, such as titration of fluorescent antibody, combined with quantitative cluster analysis, and it thereby circumvents the problem of cluster artifacts generated by overcounting of blinking fluorophores. The method was used to analyze nanocluster formation in resting and activated immune cells.

Tunable DNA Hybridization Enables Spatially and Temporally Controlled Surface-Anchoring of Biomolecular Cargo.

The controlled immobilization of biomolecules onto surfaces is relevant in biosensing and cell biological research. Spatial control is achieved by surface-tethering molecules in micro- or nanoscale patterns. Yet, there is an increasing demand for temporal control over how long biomolecular cargo stays immobilized until released into the medium. Here, we present a DNA hybridization-based approach to reversibly anchor biomolecular cargo onto micropatterned surfaces. Cargo is linked to a DNA oligonucleotide that hybridizes to a sequence-complementary, surface-tethered strand. The cargo is released from the substrate by the addition of an oligonucleotide that disrupts the duplex interaction via toehold-mediated strand displacement. The unbound tether strand can be reloaded. The generic strategy is implemented with small-molecule or protein cargo, varying DNA sequences, and multiple surface patterning routes. The approach may be used as a tool in biological research to switch membrane proteins from a locally fixed to a free state, or in biosensing to shed biomolecular receptors to regenerate the sensor surface.

Kinetic analysis of single molecule FRET transitions without trajectories.

Single molecule Förster resonance energy transfer (smFRET) is a popular tool to study biological systems that undergo topological transitions on the nanometer scale. smFRET experiments typically require recording of long smFRET trajectories and subsequent statistical analysis to extract parameters such as the states' lifetimes. Alternatively, analysis of probability distributions exploits the shapes of smFRET distributions at well chosen exposure times and hence works without the acquisition of time traces. Here, we describe a variant that utilizes statistical tests to compare experimental datasets with Monte Carlo simulations. For a given model, parameters are varied to cover the full realistic parameter space. As output, the method yields p-values which quantify the likelihood for each parameter setting to be consistent with the experimental data. The method provides suitable results even if the actual lifetimes differ by an order of magnitude. We also demonstrated the robustness of the method to inaccurately determine input parameters. As proof of concept, the new method was applied to the determination of transition rate constants for Holliday junctions.

With or without rafts? Alternative views on cell membranes.

The fundamental mechanisms of protein and lipid organization at the plasma membrane have continued to engage researchers for decades. Among proposed models, one idea has been particularly successful which assumes that sterol-dependent nanoscopic phases of different lipid chain order compartmentalize proteins, thereby modulating protein functionality. This model of membrane rafts has sustainably sparked the fields of membrane biophysics and biology, and shifted membrane lipids into the spotlight of research; by now, rafts have become an integral part of our terminology to describe a variety of cell biological processes. But is the evidence clear enough to continue supporting a theoretical concept which has resisted direct proof by observation for nearly twenty years? In this essay, we revisit findings that gave rise to and substantiated the raft hypothesis, discuss its impact on recent studies, and present alternative mechanisms to account for plasma membrane heterogeneity.