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.

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.

Phosphatidylinositol 4,5-bisphosphate (PIP2) facilitates norepinephrine transporter dimerization and modulates substrate efflux.

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 (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 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 PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.

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.

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.

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.

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.

Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy.

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophore's blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform, we reveal complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allows for robust evaluation of molecular clustering based on individually recorded single molecule localization maps.

Verifying molecular clusters by 2-color localization microscopy and significance testing.

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.

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.

Hypolipidemic effects of herbal extracts by reduction of adipocyte differentiation, intracellular neutral lipid content, lipolysis, fatty acid exchange and lipid droplet motility.

An increase in adipose tissue is caused by the increased size and number of adipocytes. Lipids accumulate in intracellular stores, known as lipid droplets (LDs). Recent studies suggest that parameters such as LD size, shape and dynamics are closely related to the development of obesity. Berberine (BBR), a natural plant alkaloid, has been demonstrated to possess anti-obesity effects. However, it remains unknown which cellular processes are affected by this compound or how effective herbal extracts containing BBR and other alkaloids actually are. For this study, we used extracts of Coptis chinensis, Mahonia aquifolium, Berberis vulgaris and Chelidonium majus containing BBR and other alkaloids and studied various processes related to adipocyte functionality. The presence of extracts resulted in reduced adipocyte differentiation, as well as neutral lipid content and rate of lipolysis. We observed that the intracellular fatty acid exchange was reduced in different LD size fractions upon treatment with BBR and Coptis chinensis. In addition, LD motility was decreased upon incubation with BBR, Coptis chinensis and Chelidonium majus extracts. Furthermore, Chelidonium majus was identified as a potent fatty acid uptake inhibitor. This is the first study that demonstrates the selected regulatory effects of herbal extracts on adipocyte function.

A micropatterning platform for quantifying interaction kinetics between the T cell receptor and an intracellular binding protein.

A complete understanding of signaling processes at the plasma membrane depends on a quantitative characterization of the interactions of the involved proteins. Fluorescence recovery after photobleaching (FRAP) is a widely used and convenient technique to obtain kinetic parameters on protein interactions in living cells. FRAP experiments to determine unbinding time constants for proteins at the plasma membrane, however, are often hampered by non-specific contributions to the fluorescence recovery signal. On the example of the interaction between the T cell receptor (TCR) and the Syk kinase ZAP70, we present here an approach based on protein micropatterning that allows the elimination of such non-specific contributions and considerably simplifies analysis of FRAP data. Specifically, detection and reference areas are created within single cells, each being either enriched or depleted in TCR, which permits the isolation of ZAP70-TCR binding in a straight-forward manner. We demonstrate the applicability of our method by comparing it to a conventional FRAP approach.

What we talk about when we talk about nanoclusters.

Superresolution microscopy results have sparked the idea that many membrane proteins are not randomly distributed across the plasma membrane but are instead arranged in nanoclusters. Frequently, these new results seemed to confirm older data based on biochemical and electron microscopy experiments. Recently, however, it was recognized that multiple countings of the very same fluorescently labeled protein molecule can be easily confused with true protein clusters. Various strategies have been developed, which are intended to solve the problem of discriminating true protein clusters from imaging artifacts. We believe that there is currently no perfect algorithm for this problem; instead, different approaches have different strengths and weaknesses. In this review, we discuss single molecule localization microscopy in view of its ability to detect nanoclusters of membrane proteins. To capture the different views on nanoclustering, we chose an unconventional style for this article: we placed its scientific content in the setting of a fictive conference, where five researchers from different fields discuss the problem of detecting and quantifying nanoclusters. Using this style, we feel that the different approaches common for different research areas can be well illustrated. Similarities to a short story by Raymond Carver are not unintentional.

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.

Direct observation of cargo transfer from HDL particles to the plasma membrane.

BACKGROUND AND AIMS: Exchange of cholesterol between high-density lipoprotein (HDL) particles and cells is a key process for maintaining cellular cholesterol homeostasis. Recently, we have shown that amphiphilic cargo derived from HDL can be transferred directly to lipid bilayers. Here we pursued this work using a fluorescence-based method to directly follow cargo transfer from HDL particles to the cell membrane. METHODS: HDL was either immobilized on surfaces or added directly to cells, while transfer of fluorescent cargo was visualized via fluorescence imaging. RESULTS: In Chinese hamster ovary (CHO) cells expressing the scavenger receptor class B type 1 (SR-B1), transfer of amphiphilic cargo from HDL particles to the plasma membrane was observed immediately after contact, whereas hydrophobic cargo remained associated with the particles; about 60% of the amphiphilic cargo of surface-bound HDL was transferred to the plasma membrane. Essentially no cargo transfer was observed in cells with low endogenous SR-B1 expression. Interestingly, transfer of fluorescently-labeled cholesterol was also facilitated by using an artificial linker to bind HDL to the cell surface. CONCLUSIONS: Our data hence indicate that the tethering function of SR-B1 is sufficient for efficient transfer of free cholesterol to the plasma membrane.

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.

Determination of the Membrane Environment of CD59 in Living Cells.

The organization and dynamics of proteins and lipids in the plasma membrane, and their role in membrane functionality, have been subject of a long-lasting debate. Specifically, it is unclear to what extent membrane proteins are affected by their immediate lipid environment and vice versa. Studies on model membranes and plasma membrane vesicles indicated preferences of proteins for lipid phases characterized by different acyl chain order; however, whether such phases do indeed exist in live cells is still not known. Here, we refine a previously developed micropatterning approach combined with single molecule tracking to quantify the influence of the glycosylphosphatidylinositol-anchored (GPI-anchored) protein CD59 on its molecular environment directly in the live cell plasma membrane. We find that locally enriched and immobilized CD59 presents obstacles to the diffusion of fluorescently labeled lipids with a different phase-partitioning behavior independent of cell cholesterol levels and type of lipid. Our results give no evidence for either specific binding of the lipids to CD59 or the existence of nanoscopic ordered membrane regions associated with CD59.

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.

Mild heat induces a distinct "eustress" response in Chinese Hamster Ovary cells but does not induce heat shock protein synthesis.

The current research on cellular heat stress management focuses on the roles of heat shock proteins (HSPs) and the proteostasis network under severe stress conditions. The mild, fever-type stress and the maintenance of membrane homeostasis are less well understood. Herein, we characterized the acute effect of mild, fever-range heat shock on membrane organization, and HSP synthesis and localization in two mammalian cell lines, to delineate the role of membranes in the sensing and adaptation to heat. A multidisciplinary approach combining ultrasensitive fluorescence microscopy and lipidomics revealed the molecular details of novel cellular "eustress", when cells adapt to mild heat by maintaining membrane homeostasis, activating lipid remodeling, and redistributing chaperone proteins. Notably, this leads to acquired thermotolerance in the complete absence of the induction of HSPs. At higher temperatures, additional defense mechanisms are activated, including elevated expression of molecular chaperones, contributing to an extended stress memory and acquired thermotolerance.

HDL particles incorporate into lipid bilayers - a combined AFM and single molecule fluorescence microscopy study.

The process, how lipids are removed from the circulation and transferred from high density lipoprotein (HDL) - a main carrier of cholesterol in the blood stream - to cells, is highly complex. HDL particles are captured from the blood stream by the scavenger receptor, class B, type I (SR-BI), the so-called HDL receptor. The details in subsequent lipid-transfer process, however, have not yet been completely understood. The transfer has been proposed to occur directly at the cell surface across an unstirred water layer, via a hydrophobic channel in the receptor, or after HDL endocytosis. The role of the target lipid membrane for the transfer process, however, has largely been overlooked. Here, we studied at the single molecule level how HDL particles interact with synthetic lipid membranes. Using (high-speed) atomic force microscopy and fluorescence correlation spectroscopy (FCS) we found out that, upon contact with the membrane, HDL becomes integrated into the lipid bilayer. Combined force and single molecule fluorescence microscopy allowed us to directly monitor the transfer process of fluorescently labelled amphiphilic lipid probe from HDL particles to the lipid bilayer upon contact.