MYC multimers shield stalled replication forks from RNA polymerase.

Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.

Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier.

Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range. In combination with genetic code expansion with unnatural amino acids and bioorthogonal click labeling with small fluorophores, photoswitching fingerprint analysis can be used advantageously to reveal information about the number of fluorophores present and their distances in the sub-10-nm range in cells.

Dynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons.

In neurons, the endoplasmic reticulum (ER) forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance; however, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high-resolution microscopy and live-cell imaging to investigate rapid movements of the ER and ribosomes in axons of cultured motoneurons after stimulation with brain-derived neurotrophic factor. Our results indicate that the ER extends into axonal growth cone filopodia, where its integrity and dynamic remodeling are regulated mainly by actin and the actin-based motor protein myosin VI (encoded by Myo6). Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with the ER in response to extracellular stimuli, which describes a novel function of axonal ER in dynamic regulation of local translation. This article has an associated First Person interview with Chunchu Deng, joint first author of the paper.

LOCAN: a python library for analyzing single-molecule localization microscopy data.

SUMMARY: Single-molecule localization microscopy has become an important part of the super-resolution microscopy toolbox in biomedical research. Software platforms for applying analytical methods to the point-based data structures are needed that offer both routine application and flexible customization of analysis procedures. We present a python library called LOCAN that consists of well-defined data structures and analysis methods for analyzing localization data in a script or computable notebook. AVAILABILITY AND IMPLEMENTATION: The package source code is released open-source under a BSD-3 license at https://github.com/super-resolution/Locan. It can be installed from the Python Package Index at https://pypi.org/project/locan. Documentation is available at https://locan.readthedocs.io.

Convex hull as diagnostic tool in single-molecule localization microscopy.

MOTIVATION: Single-molecule localization microscopy resolves individual fluorophores or fluorescence-labeled biomolecules. Data are provided as a set of localizations that distribute normally around the true fluorophore position with a variance determined by the localization precision. Characterizing the spatial fluorophore distribution to differentiate between resolution-limited localization clusters, which resemble individual biomolecules, and extended structures, which represent aggregated molecular complexes, is a common challenge. RESULTS: We demonstrate the use of the convex hull and related hull properties of localization clusters for diagnostic purposes, as a parameter for cluster selection or as a tool to determine localization precision. AVAILABILITY AND IMPLEMENTATION: https://github.com/super-resolution/Ebert-et-al-2022-supplement. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Coronaviruses Use ACE2 Monomers as Entry-Receptors.

The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1-2 receptors µm-2 . In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection, which provides SARS-CoV-2 a high infectivity.

Registration and Visualization of Correlative Super-Resolution Microscopy Data.

We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-µm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.

Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission.

γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

Nanostructure of DNA repair foci revealed by superresolution microscopy.

Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (∼200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy ( dSTORM) with a lateral resolution of ∼20 nm to analyze the focal nanostructure of the DSB marker histone γH2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained γH2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that γH2AX foci consisted of distinct circular subunits ("nanofoci") with a diameter of ∼45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that γH2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual γH2AX-containing nucleosomes. dSTORM-based γH2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of γH2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ∼0.6 and ∼1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.-Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.

ß Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice.

BACKGROUND: The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and ß-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of ß-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in ß-cells. METHODS: Mice with a floxed GC-A gene were bred to Rip-CreTG mice, thereby deleting GC-A selectively in ß-cells (ß GC-A KO). Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion were monitored in normal diet (ND)- and high-fat diet (HFD)-fed mice. ß-cell size and number were measured by immunofluorescence-based islet morphometry. RESULTS: In vitro, the insulinotropic and proliferative actions of ANP were abolished in islets isolated from ß GC-A KO mice. Concordantly, in vivo, infusion of BNP mildly enhanced baseline plasma insulin levels and glucose-induced insulin secretion in control mice. This effect of exogenous BNP was abolished in ß GC-A KO mice, corroborating the efficient inactivation of the GC-A receptor in ß-cells. Despite this under physiological, ND conditions, fasted and fed insulin levels, glucose-induced insulin secretion, glucose tolerance and ß-cell morphology were similar in ß GC-A KO mice and control littermates. However, HFD-fed ß GC-A KO animals had accelerated glucose intolerance and diminished adaptative ß-cell proliferation. CONCLUSIONS: Our studies of ß GC-A KO mice demonstrate that the cardiac hormones ANP and BNP do not modulate ß-cell's growth and secretory functions under physiological, normal dietary conditions. However, endogenous NP/GC-A signaling improves the initial adaptative response of ß-cells to HFD-induced obesity. Impaired ß-cell NP/GC-A signaling in obese individuals might contribute to the development of type 2 diabetes.

Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging.

Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.

Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition.

Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.

Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy.

The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50-60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.

Artifacts in single-molecule localization microscopy.

Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.

Dynamical fingerprints for probing individual relaxation processes in biomolecular dynamics with simulations and kinetic experiments.

There is a gap between kinetic experiment and simulation in their views of the dynamics of complex biomolecular systems. Whereas experiments typically reveal only a few readily discernible exponential relaxations, simulations often indicate complex multistate behavior. Here, a theoretical framework is presented that reconciles these two approaches. The central concept is "dynamical fingerprints" which contain peaks at the time scales of the dynamical processes involved with amplitudes determined by the experimental observable. Fingerprints can be generated from both experimental and simulation data, and their comparison by matching peaks permits assignment of structural changes present in the simulation to experimentally observed relaxation processes. The approach is applied here to a test case interpreting single molecule fluorescence correlation spectroscopy experiments on a set of fluorescent peptides with molecular dynamics simulations. The peptides exhibit complex kinetics shown to be consistent with the apparent simplicity of the experimental data. Moreover, the fingerprint approach can be used to design new experiments with site-specific labels that optimally probe specific dynamical processes in the molecule under investigation.

Super-resolution imaging of plasma membrane glycans.

Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.

PCNA as Protein-Based Nanoruler for Sub-10 nm Fluorescence Imaging.

Super-resolution microscopy has revolutionized biological imaging enabling direct insight into cellular structures and protein arrangements with so far unmatched spatial resolution. Today, refined single-molecule localization microscopy methods achieve spatial resolutions in the one-digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein-based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super-resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site-specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6 nm distances. Bioorthogonal click labeling with tetrazine-dyes and tetrazine-functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time-resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is used to resolve 6 nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super-resolution imaging techniques, particularly in complex biological environments.

Methylene blue- and thiol-based oxygen depletion for super-resolution imaging.

Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions with the aim of designing an oxygen scavenger system as an alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as ß-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere ß-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.

Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics.

Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.

Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale.

Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions, e.g. in a solvent of high viscosity, a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion, fluorescence quenching interactions, and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.