Biology subjects

Heilemann, M.

Publications and source records attributed to Heilemann, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Cell-envelope conduits enable transfer of megabase-sized double-stranded DNA between cells of the nosocomial pathogen Acinetobacter baumannii

Horizontal gene transfer in Acinetobacter baumannii has been attributed to conjugative pili, transduction, and natural transformation. Here, we describe a previously unrecognized type of chromosomal DNA exchange mediated by cell-envelope conduits that establish direct cytoplasmic continuity between neighboring A. baumannii cells. Cryo-electron tomography reveals that these conduits, which were found in multiple clinical isolates, comprise an outer membrane, a peptidoglycan layer, and an inner membrane. The ~65 nm-thick conduits contain two ~2 nm-thick filaments, which super-resolution microscopy identifies as DNA. Interstrain horizontal gene transfer assays coupled with whole-genome sequencing demonstrate the transfer and homologous recombination of chromosomal segments up to 1.1 Mbp, corresponding to as much as 27% of the A. baumannii genome. Together, these findings provide direct structural and functional evidence for a conduit-mediated large-scale chromosomal DNA exchange, thus expanding the known repertoire of horizontal gene transfer strategies that may contribute to the remarkable genomic plasticity of A. baumannii.

microbiology↗

bioRxiv2026

Quantitative mapping of nanoscale EGFR-Grb2 assemblies by DNA-PAINT

Receptor tyrosine kinase signaling is initiated by extracellular ligand binding, which drives the formation of membrane-protein assemblies that activate intracellular signal transduction. Accurately resolving the molecular composition of these assemblies in situ remains challenging due to their nanoscale dimensions and intrinsic heterogeneity. Here, we introduce a single-molecule super-resolution imaging and analysis workflow designed to resolve and quantitatively characterize individual membrane-protein assembly sites in cells. We apply this approach to the nanoscale organization of the epidermal growth factor receptor (EGFR) and its adaptor protein Grb2 following stimulation with the native ligand epidermal growth factor (EGF). As activation progresses, we observe a reduction in EGFR density at the plasma membrane, a progressive accumulation of Grb2 at EGFR assembly sites, and an increase in both dimeric and higher-order oligomeric EGFR. The experimental and analytical framework presented here is broadly applicable to the study of diverse membrane-protein assemblies.

biophysics↗

bioRxiv2026

Wash-Free Multi-Target Super-Resolution Microscopy with Photocaged DNA Labels

Super-resolution microscopy with DNA-fluorophore labels is primed for multi-target imaging of cell biological samples. However, direct interaction with the sample is required to exchange or add DNA-fluorophore labels in each imaging round, which can impair the accuracy of the imaging data at the nanometer scale. To bypass this requirement, we introduce a wash-free method that employs DNA oligonucleotides equipped with photocaging groups. Irradiation with light removes these photo-modulatable groups and changes the hybridization properties of DNA labels, enabling light-modulated targeting. We demonstrate this concept by imaging various cellular targets with confocal microscopy, single-molecule localization microscopy, and stimulated emission depletion (STED) microscopy.

biophysics↗

bioRxiv2025

Quantitative STED microscopy with DNA-fluorophore labels

Stimulated emission depletion (STED) microscopy enables super-resolution imaging of complex biological samples in 3D, in large volumes, and live. However, molecular quantification with STED has remained underexplored. Here, we present a straightforward approach for quantitative STED that enables molecule counting. For this purpose, we designed DNA-fluorophore labels that enable signal amplification and allow for reliable intensity-based quantitative imaging. We demonstrate accurate molecule counting on DNA origami. Furthermore, we visualized and quantified EGF receptor monomers and dimers in cells. In summary, we introduce a robust, fast, and easy-to-implement tool for quantitative STED microscopy with single-protein resolution.

biophysics↗

bioRxiv2025

Nanoscale spatial-omics via contrastive embedding of single-molecule localisation data

Omics approaches have revolutionised biology, and cells can now be routinely characterised on the genomic, transcriptomic and proteomic levels. However, there is an additional pillar; the (nanoscale) spatial organisation of molecules in the cell - information now accessible through super-resolution microscopy. We present a contrastive learning framework for nanoscale spatial-omics that embeds single-molecule localisation microscopy data into a latent space representing protein architecture directly to enabling comparative analysis. Using simulated and experimental data, we demonstrate its ability to enable new bioanalysis capabilities including assessing changes to cellular nanoscale architecture arising from pharmacological treatments, cell type, fluorophore selection or data-processing workflows. The approach supports downstream tasks such as clustering proteins by nanoscale organisation, mapping dose-response trajectories and identifying batch effects in replicate datasets, establishing contrastive learning as a scalable foundation for nanoscale spatial-omics and providing a platform for comparative phenotyping, quality control, and hypothesis generation.

bioinformatics↗

bioRxiv2025

Fast, Bright and Reversible Rhodamine Tags for Live-Cell Imaging

We present Rho-tag and SiR-tag, engineered protein tags derived from bacterial multidrug-resistance proteins that bind unsubstituted (silicon-) rhodamines with nanomolar affinity, enabling fast, reversible, and fluorogenic protein labeling. In live cells, Rho-tag labeling occurs within seconds -- faster than HaloTag7 -- and the tags are compatible with super-resolution methods like STED, SMLM, and MINFLUX. The high specificity of Rho-tag and SiR-tag for unsubstituted rhodamines allows their use alongside HaloTag7 and SNAP-tag. In vivo applications are demonstrated by efficient neuronal labeling in zebrafish larvae.

bioengineering↗

bioRxiv2025

Single-molecule FRET-tracking of InlB-activated MET receptors in living cells

The activation of transmembrane receptors through the binding of external ligands initiates information transfer across the cell membrane. Understanding these processes requires observations in living cells. Given the heterogeneity and lack of synchronization of such events, single-molecule experiments are required to resolve distinct sub-populations. Here, single-molecule FRET microscopy and single-particle tracking are combined to track the ligand-induced dimerization and activation of the MET receptor tyrosine kinase in the plasma membrane of living cells. First, using fluorophore-labeled variants of the MET ligand internalin B (InlB), the lifetime of a ligand-activated dimeric (MET:InlB)2 receptor complex is determined to be around 1 second. Next, diffusion coefficients of monomeric and dimeric MET:InlB complexes are extracted from single-molecule FRET trajectories, revealing an approximately 1.6-fold slower diffusion of the dimeric receptor compared to the monomeric receptor, accompanied by spatially restricted motion. The combination of single-molecule FRET and single-particle tracking provides essential biophysical parameters of membrane receptor activation in living cells.

biophysics↗

bioRxiv2025

One-click image reconstruction in single-molecule localization microscopy via deep learning

Deep neural networks have led to significant advancements in microscopy image generation and analysis. In single-molecule localization based super-resolution microscopy, neural networks are capable of predicting fluorophore positions from high-density emitter data, thus reducing acquisition time, and increasing imaging throughput. However, neural network-based solutions in localization microscopy require intensive human intervention and computation expertise to address the compromise between model performance and its generalization. For example, researchers manually tune parameters to generate training images that are similar to their experimental data; thus, for every change in the experimental conditions, a new training set should be manually tuned, and a new model should be trained. Here, we introduce AutoDS and AutoDS3D, two software programs for reconstruction of single-molecule super-resolution microscopy data that are based on Deep-STORM and DeepSTORM3D, that significantly reduce human intervention from the analysis process by automatically extracting the experimental parameters from the imaging raw data. In the 2D case, AutoDS selects the optimal model for the analysis out of a set of pre-trained models, hence, completely removing user supervision from the process. In the 3D case, we improve the computation efficiency of DeepSTORM3D and integrate the lengthy workflow into a graphic user interface that enables image reconstruction with a single click. Ultimately, we demonstrate superior performance of both pipelines compared to Deep-STORM and DeepSTORM3D for single-molecule imaging data of complex biological samples, while significantly reducing the manual labor and computation time.

bioengineering↗

bioRxiv2025

Glyco-STORM: Nanoscale Mapping of the Cellular Glycosylation Landscape

Glycosylation is a crucial biochemical modification of proteins and other biomolecules in cells that generates an exceptional structural and functional diversity. Aberrant glycosylation is implicated in numerous diseases, including neurodegenerative disorders and cancer. Despite its significance, methodological constraints to date have limited the exploration of the nanometer scale spatial arrangement of glycans across entire cells. We developed Glyco-STORM, a super-resolution imaging approach that generates nano-structural maps of cellular glycosylation. Glyco-STORM employs fluorophore-labeled lectins and multiplexed single-molecule super-resolution microscopy, in combination with nanoscale spatial pattern analysis. For example, Glyco-STORM unraveled nanodomains within the endoplasmic reticulum, subdomains along the Golgi axes, and a polarized lysosomal clathrin coat. At synaptic contact sites, mature glycans delineate the synaptic cleft and subsynaptic tubules adjacent to the postsynaptic density. In summary, Glyco-STORM elucidates the spatial arrangement of glycosylation sites from subcellular to molecular levels, revealing the previously obscured glycosylation landscape at nanoscale and establishing a spatial glycosylation code that provides a unique perspective on cellular organization distinct from traditional protein-centric views.

cell biology↗

bioRxiv2024

Smart 3D super-resolution microscopy reveals the architecture of the RNA scaffold in a nuclear body

Small subcellular organelles orchestrate key cellular functions. How biomolecules are spatially organized within these assemblies is poorly understood. Here, we report an automated super- resolution imaging and analysis workflow that integrates confocal microscopy, morphological object screening, targeted 3D super-resolution STED microscopy and quantitative image analysis. Using this smart microscopy workflow, we targeted the 3D organization of an architectural RNA that constitutes the structural backbone of paraspeckles, a membraneless nuclear organelle. Using site-specific labeling, morphological sorting and particle averaging, we reconstructed the morphological space of paraspeckles along their development cycle from over 10,000 individual particles. Applying spherical harmonics analysis, we report so-far unknown heterotypes of RNA organization. By integrating multi- positional labeling, we determined the coarse conformation of the RNA within the organelle and found the 3 end forming a loop-like structure at the surface of the paraspeckle. Our study reveals key structural features of nuclear paraspeckle structure and growth, as well as on the molecular organization of the scaffold RNA.

biophysics↗

bioRxiv2024

Localization of Albumin with Correlative Super Resolution Light- and Electron Microscopy in the Kidney

The functioning of vertebrate life relies on renal filtration of surplus fluid and elimination of low-molecular-weight waste products, while keeping serum proteins in the blood. In disease, however, there is leak of serum proteins and tracing them to identify the leaking position within tissue with a nanometer resolution poses a significant challenge. Correlative microscopy integrates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Using chemical tagging of albumin with synthetic fluorophores we achieve protein-specific labeling that preserve their post-embedding fluorescence after high-pressure freezing and freeze-substitution of murine kidney tissue. Using advanced registration techniques for super-resolution correlative light and electron microscopy, we can localize the labeled albumin with a high precision in the x-y plane of electron micrographs and cartograph its distribution. Thereby we can quantify the albumin concentration and measure a linear reduction gradient across the kidney filtration barrier. Our study shows the feasibility of combining different microscopy contrasts for tracing fluorescently labeled protein markers with super resolution in various tissue samples and opens new perspectives for correlative imaging in volume electron microscopy.

biophysics↗

bioRxiv2024

Rapid depletion and super-resolution microscopy reveal an unexpected role of the nuclear-speckle protein SRSF5 in paraspeckle assembly and dynamics during cellular stress

Nuclear speckles (NS) and paraspeckles (PS) are adjacent condensates with distinct protein composition, with serine-arginine-rich splicing factors (SRSFs) concentrated in NS. Surprisingly, we find that SRSF5 is present in both. Combining super-resolution imaging, proximity proteomics and iCLIP, we show that SRSF5 binds with PS core proteins to the PS-scaffold RNA NEAT1 and locates between PS spheres. Acute SRSF5 depletion results in reduced PS with differently packaged NEAT1. Under stress, SRSF5s association with PS increases, and without SRSF5, PS cluster assembly is impaired. Interfering with binding to purine-rich RNAs even causes PS-NS fusion. In an intriguing over-compensation, longer SRSF5 depletion reduces TDP-43 levels via premature polyadenylation, leading to NEAT1 isoform switching and more PS. We propose that SRSF5 forms a stress-specific PS shell and acts as a glue for PS clusters. Additionally, we uncover SRSF5 as a novel regulator of TDP-43 and demonstrate how acute depletion distinguishes direct from compensatory effects. HighlightsO_LINS protein SRSF5 associates with PS shells and enriches between PS spheres C_LIO_LISRSF5 binds the PS-scaffold RNA NEAT1 and ensures proper NEAT1 packaging C_LIO_LISRSF5 association with PS increases under stress and promotes cluster formation C_LIO_LIInterfering with binding to purine-rich RNAs causes the fusion of PS and NS C_LIO_LIAcute SRSF5 depletion reveals compensatory effects on PS assembly and dynamics C_LIO_LISRSF5 regulates TDP-43 levels via premature polyadenylation C_LI

cell biology↗

bioRxiv2024

Fast and long-term super-resolution imaging of ER nano-structural dynamics in living cells using a neural network

Stimulated emission depletion (STED) microscopy is a super-resolution technique that surpasses the diffraction limit and has contributed to the study of dynamic processes in living cells. However, high laser intensities induce fluorophore photobleaching and sample phototoxicity, limiting the number of fluorescence images obtainable from a living cell. Here, we address these challenges by using ultra-low irradiation intensities and a neural network for image restoration, enabling extensive imaging of single living cells. The endoplasmic reticulum (ER) was chosen as the target structure due to its dynamic nature over short and long timescales. The reduced irradiation intensity combined with denoising permitted continuous ER dynamics observation in living cells for up to 7 hours with a temporal resolution of seconds. This allowed for quantitative analysis of ER structural features over short (seconds) and long (hours) timescales within the same cell, and enabled fast 3D live-cell STED microscopy. Overall, the combination of ultra-low irradiation with image restoration enables comprehensive analysis of organelle dynamics over extended periods in living cells.

biophysics↗

bioRxiv2024

Long-term single-molecule tracking in living cells using weak-affinity protein labeling

Single-particle tracking (SPT) has become a powerful tool to monitor the dynamics of membrane proteins in living cells. However, permanent labeling strategies for SPT suffer from photobleaching as a major limitation, restricting observation times, and obstructing the study of long-term cellular processes within single living cells. Here, we use exchangeable HaloTag Ligands (xHTLs) as an easy-to-apply labeling approach for live-cell SPT and demonstrate extended observation times of individual live cells of up to 30 minutes. Using the xHTL/HT7 labeling system, we measure the ligand-induced activation kinetics of the epidermal growth factor receptor (EGFR) in single living cells. Furthermore, we generate spatial maps of receptor diffusion in cells, report non-uniform distributions of receptor activation, and the formation of spatially confined hot spots of EGFR activation. This approach represents a general strategy to monitor protein dynamics in a functional context and for extended observation times in single living cells.

biophysics↗

bioRxiv2024

Self-quenched fluorophore-DNA labels for super-resolution fluorescence microscopy

Protein labeling through transient and repetitive hybridization of short, fluorophore-labeled DNA oligonucleotides has become widely applied in various optical super-resolution microscopy methods. The main advantages are multi-target imaging and molecular quantification. A challenge is the high background signal originating from the presence of unbound fluorophore-DNA labels in solution. Here, we report self-quenching of fluorophore dimers conjugated to DNA oligonucleotides as a general concept to reduce the fluorescence background. Upon hybridization, the fluorescence signal of both fluorophores is fully restored. Here, we expand the toolbox of fluorophores suitable for self-quenching and report their spectra and hybridization equilibria. We apply self-quenched fluorophore-DNA labels to stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM) and report improved imaging performances.

biophysics↗

bioRxiv2023

Single-molecule FRET and molecular dynamics simulations reveal early activation steps of MET receptor by Listeria monocytogenes

The assembly of membrane receptors into signaling complexes is at the origin of key cellular events. Yet, we often lack detailed structural mechanistic understanding. Receptors are embedded into a complex cellular membrane, which defines their dynamics but also complicates their experimental characterizations significantly. Here, we showcase an integrative structural biology approach to investigate the activation mechanism of the human growth factor receptor MET. MET is a receptor tyrosine kinase involved in cell proliferation, migration, and survival. MET is also hijacked by the intracellular pathogen Listeria monocytogenes. Its invasion protein, internalin B (InlB), binds to MET and promotes the formation of a signaling dimer that triggers the internalization of the pathogen. Crystallography had suggested two different 2:2 MET:InlB complexes. Here, we use a combination of structural biology, modeling, molecular dynamics simulations, and in situ single-molecule Forster resonance energy transfer (smFRET) to elucidate the early events in MET activation. Simulations show that InlB binding stabilizes MET in a conformation that promotes dimer formation. smFRET identifies the organization of the in situ signaling dimer, which resembles one of the two crystal structures yet shows differences. Further MD simulations resulted in a refinement of the dimer model, which is in quantitative agreement with smFRET results. We accurately describe the structural dynamics underpinning an important cellular event and introduce a powerful methodological pipeline applicable to studying the activation of other plasma membrane receptors in situ.

biophysics↗

bioRxiv2023

The molecular architecture of the kidney slit diaphragm

Vertebrate life depends on renal function to filter excess fluid and remove low-molecular-weight waste products. An essential component of the kidney filtration barrier is the slit diaphragm (SD), a specialized cell-cell junction between podocytes. Although the constituents of the SD are largely known, its molecular organization remains elusive. Here, we use super-resolution correlative light and electron microscopy to quantify a linear rate of reduction in albumin concentration across the filtration barrier. Next, we use cryo-electron tomography of vitreous lamellae from high-pressure frozen native glomeruli to analyze the molecular architecture of the SD. The resulting densities resemble a fishnet pattern. Fitting of Nephrin and Neph1, the main constituents of the SD, results in a complex interaction pattern with multiple contact sites between the molecules. Using molecular dynamics flexible fitting, we construct a blueprint of the SD, where we describe all interactions. Our architectural understanding of the SD reconciles previous findings and provides a mechanistic framework for the development of novel therapies to treat kidney dysfunction.

cell biology↗

bioRxiv2023

Transertion and cell geometry organize the Escherichia coli nucleoid during rapid growth

AO_SCPLOWBSTRACTC_SCPLOWBacterial chromosomes are spatiotemporally organized and sensitive to environmental changes. However, the mechanisms underlying chromosome configuration and reorganization are currently not fully understood. Using 3D single-molecule localization microscopy and live-cell imaging, we show that the Escherichia coli nucleoid adopts a condensed, membrane-associated configuration during rapid growth. To study the influence of different biosynthetic processes on nucleoid morphology and positioning, we recorded multi-colour super-resolution images during drug treatment. After developing analysis routines for confocal and super-resolution images, we captured highly resolved snapshots which revealed the complete loss of the membrane-bound state of the nucleoid within 10 minutes of halting transcription and translation. This indicates an active role of transertion (coupled transcription, translation and membrane insertion) in nucleoid organization. In contrast, cell wall synthesis inhibition only affects nucleoid organization during morphological changes. Further, we provide evidence that the E. coli nucleoid spatially correlates with MreB in unperturbed E. coli cells, while this correlation diminishes in cells with changed cell geometry or upon inhibition of protein biosynthesis. Replication inhibition experiments, as well as multi-drug treatments highlight the role of entropic effects and transcription in nucleoid condensation and positioning. In summary, we provide experimental evidence for transertion as a principal organiser of the bacterial nucleoid, and show that an altered metabolic state and antibiotic treatment both lead to major changes in the degree of transertion and overall spatial organization of the nucleoid. Our high-resolution characterization reveals dynamics of antibiotic action and provides tools to quantify bacterial chromosome organization. This does not only provide valuable insights into the role of transertion, but can also be applied to study other cell-biological processes.

microbiology↗