Antigen Delivery Controlled by an On-Demand Photorelease.

To eliminate infected and cancerous cells, antigen processing and presentation play a pivotal role through the recognition of antigenic peptides displayed on Major Histocompatibility Complex class I (MHC I) molecules. Here, we developed a photostimulated antigen release system that enables the temporal inception of antigen flux. Simple and effective photocaging of the human immunodeficiency virus (HIV)-Nef73-derived epitope, a representative high-affinity MHC I ligand, was provided by steric hindrance to block the recognition by the transporter associated with antigen processing (TAP) in the peptide loading complex (PLC). In response to light, a heteronomous release of antigens and subsequent translocation in various scenarios is demonstrated, including a TAP-related ATP-binding cassette (ABC) transporter reconstituted in liposomes and the native PLC in the endoplasmic reticulum (ER) membrane of human cells. The photochemically induced 'burst' of antigens opens new opportunities for a mechanistic analysis of the antigen translocation machinery and will help to provide insights into antigen processing pathways via an on-demand, subcellular pulse-chase release of antigens.

Force-tuned avidity of spike variant-ACE2 interactions viewed on the single-molecule level.

Recent waves of COVID-19 correlate with the emergence of the Delta and the Omicron variant. We report that the Spike trimer acts as a highly dynamic molecular caliper, thereby forming up to three tight bonds through its RBDs with ACE2 expressed on the cell surface. The Spike of both Delta and Omicron (B.1.1.529) Variant enhance and markedly prolong viral attachment to the host cell receptor ACE2, as opposed to the early Wuhan-1 isolate. Delta Spike shows rapid binding of all three Spike RBDs to three different ACE2 molecules with considerably increased bond lifetime when compared to the reference strain, thereby significantly amplifying avidity. Intriguingly, Omicron (B.1.1.529) Spike displays less multivalent bindings to ACE2 molecules, yet with a ten time longer bond lifetime than Delta. Delta and Omicron (B.1.1.529) Spike variants enhance and prolong viral attachment to the host, which likely not only increases the rate of viral uptake, but also enhances the resistance of the variants against host-cell detachment by shear forces such as airflow, mucus or blood flow. We uncover distinct binding mechanisms and strategies at single-molecule resolution, employed by circulating SARS-CoV-2 variants to enhance infectivity and viral transmission.

Dynamic in Situ Confinement Triggers Ligand-Free Neuropeptide Receptor Signaling.

Membrane receptor clustering is fundamental to cell-cell communication; however, the physiological function of receptor clustering in cell signaling remains enigmatic. Here, we developed a dynamic platform to induce cluster formation of neuropeptide Y2 hormone receptors (Y2R) in situ by a chelator nanotool. The multivalent interaction enabled a dynamic exchange of histidine-tagged Y2R within the clusters. Fast Y2R enrichment in clustered areas triggered ligand-independent signaling as determined by an increase in cytosolic calcium and cell migration. Notably, the calcium and motility response to ligand-induced activation was amplified in preclustered cells, suggesting a key role of receptor clustering in sensitizing the dose response to lower ligand concentrations. Ligand-independent versus ligand-induced signaling differed in the binding of arrestin-3 as a downstream effector, which was recruited to the clusters only in the presence of the ligand. This approach allows in situ receptor clustering, raising the possibility to explore different receptor activation modalities.

Light control of the peptide-loading complex synchronizes antigen translocation and MHC I trafficking.

Antigen presentation via major histocompatibility complex class I (MHC I) molecules is essential to mount an adaptive immune response against pathogens and cancerous cells. To this end, the transporter associated with antigen processing (TAP) delivers snippets of the cellular proteome, resulting from proteasomal degradation, into the ER lumen. After peptide loading and editing by the peptide-loading complex (PLC), stable peptide-MHC I complexes are released for cell surface presentation. Since the process of MHC I trafficking is poorly defined, we established an approach to control antigen presentation by introduction of a photo-caged amino acid in the catalytic ATP-binding site of TAP. By optical control, we initiate TAP-dependent antigen translocation, thus providing new insights into TAP function within the PLC and MHC I trafficking in living cells. Moreover, this versatile approach has the potential to be applied in the study of other cellular pathways controlled by P-loop ATP/GTPases.

Photoinduced receptor confinement drives ligand-independent GPCR signaling.

Cell-cell communication relies on the assembly of receptor-ligand complexes at the plasma membrane. The spatiotemporal receptor organization has a pivotal role in evoking cellular responses. We studied the clustering of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and established a photoinstructive matrix with ultrasmall lock-and-key interaction pairs to control lateral membrane organization of hormone neuropeptide Y2 receptors in living cells by light. Within seconds, receptor clustering was modulated in size, location, and density. After in situ confinement, changes in cellular morphology, motility, and calcium signaling revealed ligand-independent receptor activation. This approach may enhance the exploration of mechanisms in cell signaling and mechanotransduction.

Probing fibronectin adsorption on chemically defined surfaces by means of single molecule force microscopy.

Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) and a quartz crystal microbalance (QCM) were respectively employed to probe interfacial characteristics of fibronectin fragment FNIII8-14 and full-length fibronectin (FN) on CH3-, OH-, COOH-, and NH2-terminated alkane-thiol self-assembled monolayers (SAMs). Force-distance curves acquired between hexahistidine-tagged FNIII8-14 immobilised on trisNTA-Ni2+ functionalized AFM cantilevers and the OH and COOH SAM surfaces were predominantly 'loop-like' (76% and 94% respectively), suggesting domain unfolding and preference for 'end-on' oriented binding, while those generated with NH2 and CH3 SAMs were largely 'mixed type' (81% and 86%, respectively) commensurate with unravelling and desorption, and 'side-on' binding. Time-dependent binding of FN to SAM-coated QCM crystals occurred in at least two phases: initial rapid coverage over the first 5 min; and variably diminishing adsorption thereafter (5-70 min). Loading profiles and the final hydrated surface concentrations reached (~ 950, ~ 1200, ~ 1400, ~ 1500 ng cm-2 for CH3, OH, COOH and NH2 SAMs) were consistent with: space-filling 'side-on' orientation and unfolding on CH3 SAM; greater numbers of FN molecules arranged 'end-on' on OH and especially COOH SAMs; and initial 'side-on' contact, followed by either (1) gradual tilting to a space-saving 'end-on' configuration, or (2) bi-/multi-layer adsorption on NH2 SAM.

Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM.

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

Multivalent Chelators for In†Vivo Protein Labeling.

With the advent of single-molecule methods, chemoselective and site-specific labeling of proteins evolved to become a central aspect in chemical biology as well as cell biology. Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. Generic methods that do not interfere with the function, dynamics, subcellular localization of proteins, and crosstalk with other factors are crucial to probe and image proteins in vitro and in living cells. Alternatives to enzyme-based tags or autofluorescent proteins are short peptide-based recognition tags. These tags provide high specificity, enhanced binding rates, bioorthogonality, and versatility. Here, we report on recent applications of multivalent chelator heads, recognizing oligohistidine-tagged proteins. The striking features of this system has facilitated the analysis of protein complexes by single-molecule approaches.

Tris -N-Nitrilotriacetic Acid Fluorophore as a Self-Healing Dye for Single-Molecule Fluorescence Imaging.

The photostability of fluorescent labels comprises one of the main limitations in single-molecule fluorescence (SMF) and super-resolution imaging. An attractive strategy to increase the photostability of organic fluorophores relies on their coupling to photostabilizers, e.g., triplet excited state quenchers, rendering self-healing dyes. Herein we report the self-healing properties of trisNTA-Alexa647 fluorophores (NTA, N-nitrilotriacetic acid). Primarily designed to specifically label biomolecules containing an oligohistidine tag, we hypothesized that the increased effective concentration of Ni(II) triplet state quenchers would lead to their improved photostability. We evaluated photon output, survival time, and photon count rate of different Alexa647-labeled trisNTA constructs differing in the length and rigidity of the fluorophore- trisNTA linker. Maximum photon output enhancements of 25-fold versus Alexa647-DNA were recorded for a short tetraproline linker, superseding the solution based photostabilization by Ni(II). Steady-state and time-resolved studies illustrate that trisNTA self-healing role is associated with a dynamic excited triplet state quenching by Ni(II). Here improved photophysical/photochemical properties require for a judicious choice of linker length and rigidity, and in turn a balance between rapid dynamic triplet excited state quenching versus dynamic/static singlet excited state quenching. TrisNTA fluorophores offer superior properties for SMF allowing specific labeling and increased photostability, making them ideal candidates for extended single-molecule imaging techniques.

Interactions of bacteriophage T4 adhesin with selected lipopolysaccharides studied using atomic force microscopy.

The interaction between the T4 bacteriophage gp37 adhesin and the bacterial lipopolysaccharide (LPS) is a well-studied system, however, the affinity and strength of the interaction haven't been analyzed so far. Here, we use atomic force microscopy to determine the strength of the interaction between the adhesin and its receptor, namely LPS taken from a wild strain of E. coli B. As negative controls we used LPSs of E. coli O111:B and Hafnia alvei. To study the interaction an AFM tip modified with the gp37 adhesin was used to scan surfaces of mica covered with one of the three different LPSs. Using the correlation between the surface topography images and the tip-surface interaction we could verify the binding between the specific LPS and the tip in contrast to the very weak interaction between the tip and the non-binding LPSs. Using force spectroscopy we could then measure the binding strength by pulling on the AFM tip until it lifted off from the surface. The force necessary to break the interaction between gp37 and LPS from E. coli B, LPS from E. coli O111:B and LPS from H. alvei were measured to be 70 ± 29 pN, 46 ± 13 pN and 45 ± 14 pN, respectively. The latter values are likely partially due to non-specific interaction between the gp37 and the solid surface, as LPS from E. coli O111:B and LPS from H. alvei have been shown to not bind to gp37, which is confirmed by the low correlation between binding and topography for these samples.

Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.

With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the 'labeling barrier' and to bypass photobleaching in multi-plane, whole-cell 3D experiments.

Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA.

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3' termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3' terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3' vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.

EB1 interacts with outwardly curved and straight regions of the microtubule lattice.

EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice. Microtubules assembled in the presence of GTP, different GTP analogues or cell extracts display similarly curved sheets at their growing ends, which gradually straighten as their protofilament number increases until they close into a tube. Together, our data provide unique structural information on the interaction of EB1 with growing microtubule ends. They further offer insights into the conformational changes that tubulin dimers undergo during microtubule assembly and the architecture of the GTP-cap region.

Live-cell protein labelling with nanometre precision by cell squeezing.

Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼ 1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy.

Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM.

A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

Mechanistic Basis for Epitope Proofreading in the Peptide-Loading Complex.

The peptide-loading complex plays a pivotal role in Ag processing and is thus central to the efficient immune recognition of virally and malignantly transformed cells. The underlying mechanism by which MHC class I (MHC I) molecules sample immunodominant peptide epitopes, however, remains poorly understood. In this article, we delineate the interaction between tapasin (Tsn) and MHC I molecules. We followed the process of peptide editing in real time after ultra-fast photoconversion to pseudoempty MHC I molecules. Tsn discriminates between MHC I loaded with optimal and MHC I bound to suboptimal cargo. This differential interaction is key to understanding the kinetics of epitope proofreading. To elucidate the underlying mechanism at the atomic level, we modeled the Tsn/MHC I complex using all-atom molecular dynamics simulations. We present a catalytic working cycle, in which Tsn binds to MHC I with suboptimal cargo and thereby adjusts the energy landscape in favor of MHC I complexes with immunodominant epitopes.

SLAP: Small Labeling Pair for Single-Molecule Super-Resolution Imaging.

Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small-molecule recognition unit (Ni-trisNTA) and the genetically encoded minimal protein His6-10 -tag. High-density protein tracing by SLAP was demonstrated. This technique allows super-resolution fluorescence imaging and fulfills the necessary sampling criteria for single-molecule localization-based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).

His-tagged norovirus-like particles: A versatile platform for cellular delivery and surface display.

In addition to vaccines, noninfectious virus-like particles (VLPs) that mimic the viral capsid show an attractive possibility of presenting immunogenic epitopes or targeting molecules on their surface. Here, functionalization of norovirus-derived VLPs by simple non-covalent conjugation of various molecules is shown. By using the affinity between a surface-exposed polyhistidine-tag and multivalent tris-nitrilotriacetic acid (trisNTA), fluorescent dye molecules and streptavidin-biotin conjugated to trisNTA are displayed on the VLPs to demonstrate the use of these VLPs as easily modifiable nanocarriers as well as a versatile vaccine platform. The VLPs are able to enter and deliver surface-displayed fluorescent dye into HEK293T cells via a surface-attached cell internalization peptide (VSV-G). The ease of manufacturing, the robust structure of these VLPs, and the straightforward conjugation provide a technology, which can be adapted to various applications in biomedicine.

A negative feedback modulator of antigen processing evolved from a frameshift in the cowpox virus genome.

Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.