Glycan-directed SARS-CoV-2 inhibition by leek extract and lectins with insights into the mode-of-action of Concanavalin A.

Four years after its outbreak, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global challenge for human health. At its surface, SARS-CoV-2 features numerous extensively glycosylated spike proteins. This glycan coat supports virion docking and entry into host cells and at the same time renders the virus less susceptible to neutralizing antibodies. Given the high genetic plasticity of SARS-CoV-2 and the rapid emergence of immune escape variants, targeting the glycan shield by carbohydrate-binding agents emerges as a promising strategy. However, the potential of carbohydrate-targeting reagents as viral inhibitors remains underexplored. Here, we tested seven plant-derived carbohydrate-binding proteins, called lectins, and one crude plant extract for their antiviral activity against SARS-CoV-2 in two types of human lung cells: A549 cells ectopically expressing the ACE2 receptor and Calu-3 cells. We identified three lectins and an Allium porrum (leek) extract inhibiting SARS-CoV-2 infection in both cell systems with selectivity indices (SI) ranging between >2 and >299. Amongst these, the lectin Concanavalin A (Con A) exerted the most potent and broad activity against a panel of SARS-CoV-2 variants. We used multiplex super-resolution microscopy to address lectin interactions with SARS-CoV-2 and its host cells. Notably, we discovered that Con A not only binds to SARS-CoV-2 virions and their host cells, but also causes SARS-CoV-2 aggregation. Thus, Con A exerts a dual mode-of-action comprising both, antiviral and virucidal, mechanisms. These results establish Con A and other plant lectins as candidates for COVID-19 prevention and basis for further drug development.

Molecular basis of JAK2 activation in erythropoietin receptor and pathogenic JAK2 signaling.

Janus kinase 2 (JAK2) mediates type I/II cytokine receptor signaling, but JAK2 is also activated by somatic mutations that cause hematological malignancies by mechanisms that are still incompletely understood. Quantitative superresolution microscopy (qSMLM) showed that erythropoietin receptor (EpoR) exists as monomers and dimerizes upon Epo stimulation or through the predominant JAK2 pseudokinase domain mutations (V617F, K539L, and R683S). Crystallographic analysis complemented by kinase activity analysis and atomic-level simulations revealed distinct pseudokinase dimer interfaces and activation mechanisms for the mutants: JAK V617F activity is driven by dimerization, K539L involves both increased receptor dimerization and kinase activity, and R683S prevents autoinhibition and increases catalytic activity and drives JAK2 equilibrium toward activation state through a wild-type dimer interface. Artificial intelligence-guided modeling and simulations revealed that the pseudokinase mutations cause differences in the pathogenic full-length JAK2 dimers, particularly in the FERM-SH2 domains. A detailed molecular understanding of mutation-driven JAK2 hyperactivation may enable novel therapeutic approaches to selectively target pathogenic JAK2 signaling.

Channelrhodopsin-2 Oligomerization in Cell Membrane Revealed by Photo-Activated Localization Microscopy.

Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/µm2 . Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

The function of ER-phagy receptors is regulated through phosphorylation-dependent ubiquitination pathways.

Selective autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is an important regulator of ER remodeling and essential to maintain cellular homeostasis during environmental changes. We recently showed that members of the FAM134 family play a critical role during stress-induced ER-phagy. However, the mechanisms on how they are activated remain largely unknown. In this study, we analyze phosphorylation of FAM134 as a trigger of FAM134-driven ER-phagy upon mTOR (mechanistic target of rapamycin) inhibition. An unbiased screen of kinase inhibitors reveals CK2 to be essential for FAM134B- and FAM134C-driven ER-phagy after mTOR inhibition. Furthermore, we provide evidence that ER-phagy receptors are regulated by ubiquitination events and that treatment with E1 inhibitor suppresses Torin1-induced ER-phagy flux. Using super-resolution microscopy, we show that CK2 activity is essential for the formation of high-density FAM134B and FAM134C clusters. In addition, dense clustering of FAM134B and FAM134C requires phosphorylation-dependent ubiquitination of FAM134B and FAM134C. Treatment with the CK2 inhibitor SGC-CK2-1 or mutation of FAM134B and FAM134C phosphosites prevents ubiquitination of FAM134 proteins, formation of high-density clusters, as well as Torin1-induced ER-phagy flux. Therefore, we propose that CK2-dependent phosphorylation of ER-phagy receptors precedes ubiquitin-dependent activation of ER-phagy flux.

Neural network-assisted single-molecule localization microscopy with a weak-affinity protein tag.

Single-molecule localization microscopy achieves nanometer spatial resolution by localizing single fluorophores separated in space and time. A major challenge of single-molecule localization microscopy is the long acquisition time, leading to low throughput, as well as to a poor temporal resolution that limits its use to visualize the dynamics of cellular structures in live cells. Another challenge is photobleaching, which reduces information density over time and limits throughput and the available observation time in live-cell applications. To address both challenges, we combine two concepts: first, we integrate the neural network DeepSTORM to predict super-resolution images from high-density imaging data, which increases acquisition speed. Second, we employ a direct protein label, HaloTag7, in combination with exchangeable ligands (xHTLs), for fluorescence labeling. This labeling method bypasses photobleaching by providing a constant signal over time and is compatible with live-cell imaging. The combination of both a neural network and a weak-affinity protein label reduced the acquisition time up to ∼25-fold. Furthermore, we demonstrate live-cell imaging with increased temporal resolution, and capture the dynamics of the endoplasmic reticulum over extended time without signal loss.

Self-quenched Fluorophore Dimers for DNA-PAINT and STED Microscopy.

Super-resolution techniques like single-molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non-covalent, weak affinity-based transient labelling systems. DNA-based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi-target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore-labelled single-stranded oligonucleotides. Here, we introduce short-distance self-quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2-fold. We characterise the optical and thermodynamic properties of fluorophore-dimer single-stranded DNA, and show super-resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background.

DBlink: dynamic localization microscopy in super spatiotemporal resolution via deep learning.

Single-molecule localization microscopy (SMLM) has revolutionized biological imaging, improving the spatial resolution of traditional microscopes by an order of magnitude. However, SMLM techniques require long acquisition times, typically a few minutes, to yield a single super-resolved image, because they depend on accumulation of many localizations over thousands of recorded frames. Hence, the capability of SMLM to observe dynamics at high temporal resolution has always been limited. In this work, we present DBlink, a deep-learning-based method for super spatiotemporal resolution reconstruction from SMLM data. The input to DBlink is a recorded video of SMLM data and the output is a super spatiotemporal resolution video reconstruction. We use a convolutional neural network combined with a bidirectional long short-term memory network architecture, designed for capturing long-term dependencies between different input frames. We demonstrate DBlink performance on simulated filaments and mitochondria-like structures, on experimental SMLM data under controlled motion conditions and on live-cell dynamic SMLM. DBlink's spatiotemporal interpolation constitutes an important advance in super-resolution imaging of dynamic processes in live cells.

When Weak Is Strong: A Plea for Low-Affinity Binders for Optical Microscopy.

The exploitation of low-affinity molecular interactions in protein labeling is an emerging topic in optical microscopy. Such non-covalent and low-affinity interactions can be realized with various concepts from chemistry and for different molecule classes, and lead to a constant renewal of fluorescence signals at target sites. Further benefits are a versatile use across microscopy methods, in 3D, live and many-target applications. In recent years, several classes of low-affinity labels were developed and a variety of powerful applications demonstrated. Still, this research field is underdeveloped, while the potential is huge.

Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

Biased activation of the receptor tyrosine kinase HER2.

HER2 belongs to the ErbB sub-family of receptor tyrosine kinases and regulates cellular proliferation and growth. Different from other ErbB receptors, HER2 has no known ligand. Activation occurs through heterodimerization with other ErbB receptors and their cognate ligands. This suggests several possible activation paths of HER2 with ligand-specific, differential response, which has so far remained unexplored. Using single-molecule tracking and the diffusion profile of HER2 as a proxy for activity, we measured the activation strength and temporal profile in live cells. We found that HER2 is strongly activated by EGFR-targeting ligands EGF and TGFα, yet with a distinguishable temporal fingerprint. The HER4-targeting ligands EREG and NRGß1 showed weaker activation of HER2, a preference for EREG, and a delayed response to NRGß1. Our results indicate a selective ligand response of HER2 that may serve as a regulatory element. Our experimental approach is easily transferable to other membrane receptors targeted by multiple ligands.

A framework for evaluating the performance of SMLM cluster analysis algorithms.

Single-molecule localization microscopy (SMLM) generates data in the form of coordinates of localized fluorophores. Cluster analysis is an attractive route for extracting biologically meaningful information from such data and has been widely applied. Despite a range of cluster analysis algorithms, there exists no consensus framework for the evaluation of their performance. Here, we use a systematic approach based on two metrics to score the success of clustering algorithms in simulated conditions mimicking experimental data. We demonstrate the framework using seven diverse analysis algorithms: DBSCAN, ToMATo, KDE, FOCAL, CAML, ClusterViSu and SR-Tesseler. Given that the best performer depended on the underlying distribution of localizations, we demonstrate an analysis pipeline based on statistical similarity measures that enables the selection of the most appropriate algorithm, and the optimized analysis parameters for real SMLM data. We propose that these standard simulated conditions, metrics and analysis pipeline become the basis for future analysis algorithm development and evaluation.

Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy.

The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, the irreversible attachment of the probe to HaloTag precludes imaging applications that require transient binding of the probe and comes with the risk of irreversible photobleaching. Here, we introduce exchangeable ligands for fluorescence labeling of HaloTag (xHTLs) that reversibly bind to HaloTag and that can be coupled to rhodamines of different colors. In stimulated emission depletion (STED) microscopy, probe exchange of xHTLs allows imaging with reduced photobleaching as compared to covalent HaloTag labeling. Transient binding of fluorogenic xHTLs to HaloTag fusion proteins enables points accumulation for imaging in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT and STED microscopy. xHTLs thus open up new possibilities in imaging across microscopy platforms for a widely used labeling approach.

Unbiased choice of global clustering parameters for single-molecule localization microscopy.

Single-molecule localization microscopy resolves objects below the diffraction limit of light via sparse, stochastic detection of target molecules. Single molecules appear as clustered detection events after image reconstruction. However, identification of clusters of localizations is often complicated by the spatial proximity of target molecules and by background noise. Clustering results of existing algorithms often depend on user-generated training data or user-selected parameters, which can lead to unintentional clustering errors. Here we suggest an unbiased algorithm (FINDER) based on adaptive global parameter selection and demonstrate that the algorithm is robust to noise inclusion and target molecule density. We benchmarked FINDER against the most common density based clustering algorithms in test scenarios based on experimental datasets. We show that FINDER can keep the number of false positive inclusions low while also maintaining a low number of false negative detections in densely populated regions.

Synergizing Exchangeable Fluorophore Labels for Multitarget STED Microscopy.

Investigating the interplay of cellular proteins with optical microscopy requires multitarget labeling. Spectral multiplexing using high-affinity or covalent labels is limited in the number of fluorophores that can be discriminated in a single imaging experiment. Advanced microscopy methods such as STED microscopy additionally demand balanced excitation, depletion, and emission wavelengths for all fluorophores, further reducing multiplexing capabilities. Noncovalent, weak-affinity labels bypass this "spectral barrier" through label exchange and sequential imaging of different targets. Here, we combine exchangeable HaloTag ligands, weak-affinity DNA hybridization, and hydrophophic and protein-peptide interactions to increase labeling flexibility and demonstrate six-target STED microscopy in single cells. We further show that exchangeable labels reduce photobleaching as well as facilitate long acquisition times and multicolor live-cell and high-fidelity 3D STED microscopy. The synergy of different types of exchangeable labels increases the multiplexing capabilities in fluorescence microscopy, and by that, the information content of microscopy images.

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.

Nanoscale organization of the MHC I peptide-loading complex in human dendritic cells.

Dendritic cells (DCs) translate local innate immune responses into long-lasting adaptive immunity by priming antigen-specific T cells. Accordingly, there is an ample interest in exploiting DCs for therapeutic purposes, e.g., in personalized immunotherapies. Despite recent advances in elucidating molecular pathways of antigen processing, in DCs the exact spatial organization of the underlying processes is largely unknown. Here, we unraveled the nanoscale organization of the transporter associated with antigen processing (TAP)-dependent peptide-loading machinery in human monocyte-derived DCs (moDC). We detected an unexpected accumulation of MHC I peptide-loading complexes (PLCs) and TAP-dependent peptide compartmentalization in protrusions of activated DCs. Using single-molecule localization microscopy we revealed that PLCs display homogeneously sized assemblies, independent of the DC activation status or cellular localization. Our data indicate that moDCs show augmentation of subcellular PLC density during DC maturation. We observed a twofold density increase in the cell body, while an even fourfold accumulation was detected in the tips of the protrusions at the mature DC stage in comparison to immature DCs. In these tip regions, PLC assemblies are found along highly compressed tubular ER networks. These findings provide novel insights into nanoscale organization of the antigen presentation machinery, and open new perspectives on the T cell stimulatory capacity of DCs.

Fast DNA-PAINT imaging using a deep neural network.

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution technique with relatively easy-to-implement multi-target imaging. However, image acquisition is slow as sufficient statistical data has to be generated from spatio-temporally isolated single emitters. Here, we train the neural network (NN) DeepSTORM to predict fluorophore positions from high emitter density DNA-PAINT data. This achieves image acquisition in one minute. We demonstrate multi-colour super-resolution imaging of structure-conserved semi-thin neuronal tissue and imaging of large samples. This improvement can be integrated into any single-molecule imaging modality to enable fast single-molecule super-resolution microscopy.

DeepBacs for multi-task bacterial image analysis using open-source deep learning approaches.

This work demonstrates and guides how to use a range of state-of-the-art artificial neural-networks to analyse bacterial microscopy images using the recently developed ZeroCostDL4Mic platform. We generated a database of image datasets used to train networks for various image analysis tasks and present strategies for data acquisition and curation, as well as model training. We showcase different deep learning (DL) approaches for segmenting bright field and fluorescence images of different bacterial species, use object detection to classify different growth stages in time-lapse imaging data, and carry out DL-assisted phenotypic profiling of antibiotic-treated cells. To also demonstrate the ability of DL to enhance low-phototoxicity live-cell microscopy, we showcase how image denoising can allow researchers to attain high-fidelity data in faster and longer imaging. Finally, artificial labelling of cell membranes and predictions of super-resolution images allow for accurate mapping of cell shape and intracellular targets. Our purposefully-built database of training and testing data aids in novice users' training, enabling them to quickly explore how to analyse their data through DL. We hope this lays a fertile ground for the efficient application of DL in microbiology and fosters the creation of tools for bacterial cell biology and antibiotic research.

Assessing Antigen Presentation on the Surface of Plasmodium falciparum-Infected Erythrocytes by Photoactivated Localization Microscopy (PALM).

Super-resolution microscopy in the form of photoactivated localization microscopy (PALM) offers the possibility of counting single molecules in a cell, a cellular compartment or a molecular complex. PALM can, therefore, underpin molecular and biochemical processes with a numeric and stoichiometric understanding of the interacting players. Here, we introduce the physical principles underlying PALM and provide a step-by-step protocol of how to apply PALM to questions related to the biology and pathophysiology of P. falciparum and other malaria parasites.

Cyclophilin anaCyp40 regulates photosystem assembly and phycobilisome association in a cyanobacterium.

Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.