Biology subjects

Wüstner, D.

Publications and source records attributed to Wüstner, D..

12 recordsLinked to original sources

bioRxiv2026

Multimodal 3D imaging reveals a central role for lysosomes indissolution of cholesterol crystals by macrophages

Formation of cholesterol crystals (CCs) is a key event during the development of atherosclerosis, but the molecular mechanisms of their degradation within cells are poorly understood. By incorporating the fluorescent cholesterol analogue TopFluor-Cholesterol (TF-Chol) into CCs, we were able to visualize the uptake of CCs in macrophages using correlative fluorescence and soft X-ray microscopy. Using quantitative 3D live-cell imaging, we show that CCs are processed in late endosomes and lysosomes (LE/Lys), resulting in formation of TF-Chol containing lipid droplets (LDs) over time. Inhibition of lysosomal sterol export with U18666A caused accumulation of TF-Chol in LE/Lys, and inhibition of lysosomal acidification with bafilomycin A1 led to reduced dissolution of the CCs. Using a novel assay combined with 3D image processing, we show that large CCs in contact with macrophages are processed via lysosomal exocytosis followed by extracellular and intracellular degradation of CCs. Treating macrophages with a fluorescent version of cyclodextrin (CD) promoted the dissolution of CCs and enhanced the formation of LDs enriched with TF-Chol. The majority of fluorescent CD co-localized with a marker for LE/Lys during this process, suggesting that intracellular delivery to LE/Lys may contribute to the dissolution of CCs. Dehydroergosterol (DHE) is an intrinsically fluorescent sterol closely mimicking the properties and behavior of cholesterol. DHE is known to self-associate into aggregates and crystals, and by using fluorescence spectroscopy and specialized ultraviolet (UV) microscopy, we found that CD enhances the dissolution of DHE crystals in vitro and in cells. Together, our findings highlight the lysosomal pathway as responsible for dissolution of CCs, cholesterol trafficking, and efflux in macrophages.

biochemistry↗

bioRxiv2026

Analysis of motor-based transport in primary cilia by dynamic mode decomposition of live-cell imaging data

Kinesin-3 motor proteins are increasingly recognized for their important roles in cilia. The mammalian kinesin-3 motor KIF13B moves bidirectionally in primary cilia and regulates ciliary content, but its relationship to the intraflagellar transport (IFT) machinery is unclear. Here, we combine quantitative live-cell imaging with a new kymograph analysis based on dynamic mode decomposition (DMD) to separate mobile from immobile protein populations in primary cilia. This approach simplifies extraction of molecular velocities from kymographs and reveals that a KIF13B deletion mutant retaining only the motor domain and part of the forkhead-associated domain does not alter steady-state IFT velocity or frequency. However, when retrograde dynein-2 function is inhibited by Ciliobrevin D, both anterograde and retrograde IFT velocities decrease in parental cells, as expected, but remain unchanged in KIF13B mutant cells. Structured illumination, confocal, and STED microscopy further show that KIF13B localizes to the ciliary membrane and concentrates at the periciliary membrane region and the centriolar subdistal appendages, below the distal appendage marker FBF1. Our improved kymograph approach provides new insight into KIF13B ciliary function and simplifies the quantitative analysis of ciliary protein transport.

biophysics↗

bioRxiv2026

A semantic segmentation model to predict subcellular glycogen localization using transmission electron microscopy images

Transmission electron microscopy (TEM) is the gold standard for assessing subcellular glycogen localization in skeletal muscle fibres, but conventional manual analysis is extremely time-consuming and limits large-scale studies. Here, we developed and validated a deep learning-based semantic segmentation approach to automate quantification of glycogen particles across defined subcellular compartments in human skeletal muscle. Skeletal muscle biopsies were obtained from seven healthy men under conditions of normal, depleted, and supercompensated glycogen content. TEM images were acquired from myofibrillar and subsarcolemmal regions and manually annotated to train two complementary attention U-Net models: a region model identifying subcellular structures (intermyofibrillar space, intramyofibrillar regions including A-band, I-band and Z-disc, and mitochondria) and a glycogen model detecting individual glycogen particles. Combining the two models enabled estimation of compartment-specific glycogen areal densities. Model performance was evaluated against manual point-counting. At the fibre level, estimates based on 10-12 images per region achieved biases below 15% and coefficient of variation below 26% for all compartments. Importantly, model-derived total glycogen volume density showed strong concordance with biochemically determined muscle glycogen content across biopsies. In conclusion, this validated semantic segmentation workflow provides a robust, objective, and highly time-efficient tool for quantifying subcellular glycogen distribution in skeletal muscle. The model substantially reduces analysis time and enables high-throughput investigations of compartmentalized glycogen metabolism, with model weights and code made openly available.

physiology↗

bioRxiv2026

The orientation of cholesterol's hydroxyl group affects its membrane dynamics and intracellular transport

The brain, though less than 10% of body mass, contains about 25% of total cholesterol (CHL), emphasizing CHLs key role in neuronal function. Many CHL actions are stereospecific, as shown by differences from its 3-hydroxy epimer, epicholesterol (epiCHL). How this minor structural change alters membrane properties and sterol transport remains unclear. Here, we compare fluorescent analogs of CHL (cholestatrienol, CTL) and epiCHL (epicholestatrienol, epiCTL), which closely mimic their natural counterparts. Biophysical membrane properties, such as flip-flop, acyl-chain ordering, and interbilayer transfer, depend on the orientation of the 3-hydroxy group. Similarly, transport by sterol transport proteins (STPs) and intracellular trafficking of the sterols in human astrocytes are stereospecific. Treatment with 25-hydroxycholesterol increases uptake of both epimers, but only CTL shows enhanced esterification and lipid droplet storage. These findings demonstrate that subtle cholesterol structural changes affect cellular homeostasis and establish epiCTL as a useful probe of sterol stereospecificity and trafficking.

biophysics↗

bioRxiv2025

Live-cell imaging and lipidomics of low density lipoprotein containing intrinsically fluorescent cholesteryl esters

Low density lipoprotein (LDL) delivers cholesterol to cells in the body in the form of cholesteryl esters (CEs), and dysfunction of this pathway is associated with various diseases. Due to the lack of suitable tools, our understanding of the intracellular transport and hydrolysis of CEs is limited. We present a novel approach for studying LDL-derived CEs in cells, using fatty acyl chain conjugates of the intrinsically fluorescent cholestatrienol (CTL). We demonstrate that CTL esters reconstituted into LDL particles are hydrolyzed in late endosomes and lysosomes (LE/LYSs) by acid lipase, while an LDL-derived CTL ether analog cannot leave LE/LYSs. Using live-cell imaging, lipidomics, and multimodal Bayesian modeling, we discover a sequential biphasic transport of LDL-derived CTL to LE/LYSs matching the kinetics of the lysosomal hydrolysis of LDL-associated CTL-ester with a half-time of 3.0 hours. Hydrolyzed CTL derived from LDL-associated CTL esters is rapidly re-esterified with a similar half-time and stored in lipid droplets, demonstrating efficient sterol transport to the endoplasmic reticulum (ER). The latter is supported by the detection of a faint CTL staining in the ER and by extensive contact formation between endo-lysosomes containing LDL and ER tubules. Using lipidomics and Bayesian kinetic modeling, we also track LDL-derived CEs and triacylglycerols in cells and determine the uptake kinetics for each lipid species individually. Our novel approach allows for precise measurement of post-endocytic trafficking and metabolism of LDL-derived cholesterol and other lipids in living cells.

biochemistry↗

bioRxiv2024

Demonstrating Soft X-Ray Tomography in the lab for correlative cryogenic biological imaging using X-rays and light microscopy

Soft X-ray tomography (SXT) enables native-contrast three-dimensional (3D) imaging of fully hydrated, cryogenically preserved biological samples, revealing ultrastructural details without the need for staining, embedding, or sectioning. Traditionally available only at synchrotron facilities, recent advances in laser-driven plasma sources have led to the development of compact soft X-ray microscopes, such as the SXT-100. The SXT-100 achieves imaging resolutions down to 54 nm full-pitch, with tomograms acquired in 30 minutes to two hours. Integrated with an epifluorescence microscope, the SXT-100 facilitates correlative workflows by bridging fluorescence and electron microscopy while preserving the structural integrity of vitrified samples. We demonstrate the capabilities of the SXT-100 through various use cases, including imaging Euglena gracilis, Saccharomyces cerevisiae yeast cells, and nanoparticles in mammalian cells. The relatively short tomogram acquisition times, the virtually non-destructive nature of soft X-ray tomography, and its quantitative imaging capabilities underscore its potential as a powerful tool for advanced biological imaging. Future developments promise enhanced throughput and deeper integration with emerging correlative imaging modalities, and a wider variety of sample types including tissue.

biophysics↗

bioRxiv2023

Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability becomes compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and resulted in reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogs of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. We conclude that ergosterol-specific aggregation of natamycin in the yeast PM underlies its antifungal activity, which can be synergistically enhanced by inhibitors of sphingolipid synthesis. SignificanceErgosterol is the major sterol in the membranes of fungi and a major target for antifungal treatments. Polyene macrolides, such as natamycin, are known to target ergosterol but the underlying mechanisms for their preference for this yeast sterol compared to mammalian cholesterol is not understood. This study shows that natamycin forms aggregates when associated with yeast S. cerevisiae in an ergosterol-dependent manner. Cholesterol can only partially substitute for ergosterol with respect to natamycin binding and aggregation. Membrane-associated aggregation of natamycin is not the result of pre-formed sterol domains in the cell membrane, as we show by direct visualization of minimally modified ergosterol and cholesterol analogs. Inhibiting sphingolipid synthesis increased membrane association and antifungal activity of natamycin, suggesting that targeting sphingolipids in combination with polyene macrolides could lead to novel drug treatment approaches against fungal infections.

biophysics↗

bioRxiv2023

Synthesis and characterization of novel intrinsically fluorescent analogs of cholesterol with improved photophysical properties.

Live-cell imaging of cholesterol trafficking depends on suitable cholesterol analogs. However, existing fluorescent analogs of cholesterol either show very different physico-chemical properties compared to cholesterol or demand excitation in the ultraviolet spectral region. We present novel intrinsically fluorescent sterols containing four conjugated double bonds in the ring system and either a hydroxy or a keto group in the C3 position. Synthesis of these probes involves dehydrogenation of 7-dehydrocholesterol using mercury(II) acetate, Swern oxidation/dehydrogenation, and stereoselective Luche reduction. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy reveal that the analog with a 3-hydroxy-group like cholesterol can condense fatty acyl chains and form hydrogen bonds to water molecules at the bilayer interface. The emission of both probes is red-shifted by 80-120 nm compared to the widely used sterol analogs dehydroergosterol or cholestatrienol. This allows for their imaging on conventional microscopes, as we here show in giant unilamellar vesicles. These experiments reveal a preferred partitioning of both sterol probes into the biologically relevant liquid-ordered phase. In conclusion, we present a synthesis strategy leading to novel intrinsically fluorescent sterol probes with close resemblance of cholesterol. Their improved photophysical properties will allow for live-cell imaging of sterol transport in the future.

biophysics↗

bioRxiv2023

Kinetic modelling of sterol transport between plasma membrane and endo-lysosomes based on quantitative fluorescence and X-ray imaging data

Niemann Pick type C1 and C2 (NPC1 and NPC2) are two sterol-binding proteins which, together, orchestrate cholesterol transport through late endosomes and lysosomes (LE/LYSs). NPC2 can facilitate sterol exchange between model membranes severalfold, but how this is connected to its function in cells is poorly understood. Using fluorescent analogs of cholesterol and quantitative fluorescence microscopy, we have recently measured the transport kinetics of sterol between plasma membrane (PM), recycling endosomes (REs) and LE/LYSs in control and NPC2 deficient fibroblasts. Here, we employ kinetic modeling of this data to determine rate constants for sterol transport between intracellular compartments. Our model predicts that sterol is trapped in intraluminal vesicles (ILVs) of LE/LYSs in the absence of NPC2, causing delayed sterol export from LE/LYSs in NPC2 deficient fibroblasts. Using soft X-ray tomography, we confirm, that LE/LYSs of NPC2 deficient cells but not of control cells contain enlarged, carbon-rich intraluminal vesicular structures, supporting our model prediction of lipid accumulation in ILVs. By including sterol export via exocytosis of ILVs as exosomes and by release of vesicles - ectosomes - from the PM, we can reconcile measured sterol efflux kinetics and show that both pathways can be reciprocally regulated by the intraluminal sterol transfer activity of NPC2 inside LE/LYSs. Our results thereby connect the in vitro function of NPC2 as sterol transfer protein between membranes with its in vivo function.

biophysics↗

bioRxiv2022

Single-molecule tracking reveals two low-mobility states for chromatin and transcriptional regulators within the nucleus

How transcription factors (TFs) navigate the complex nuclear environment to assemble the transcriptional machinery at specific genomic loci remains elusive. Using single-molecule tracking, coupled with machine learning, we examined the mobility of multiple transcriptional regulators. We show that H2B and ten different transcriptional regulators display two distinct low-mobility states. Our results indicate that both states represent dynamic interactions with chromatin. Ligand activation results in a dramatic increase in the proportion of steroid receptors in the lowest mobility state. Mutational analysis revealed that only chromatin interactions in the lowest mobility state require an intact DNA-binding domain as well as oligomerization domains. Importantly, these states are not spatially separated as previously believed but in fact, individual H2B and TF molecules can dynamically switch between them. Together, our results identify two unique and distinct low-mobility states of transcriptional regulators that appear to represent common pathways for transcription activation in mammalian cells.

biophysics↗

bioRxiv2022

Image segmentation and separation of spectrally similar dyes in fluorescence microscopy by dynamic mode decomposition of photobleaching kinetics

BackgroundImage segmentation in fluorescence microscopy is often based on spectral separation of fluorescent probes (color-based segmentation) or on significant intensity differences in individual image regions (intensity-based segmentation). These approaches fail, if dye fluorescence shows large spectral overlap with other employed probes or with strong cellular autofluorescence. ResultsHere, a novel model-free approach is presented which determines bleaching kinetics based on dynamic mode decomposition (DMD) and uses the inferred photobleaching kinetics to distinguish different probes or dye molecules from autofluorescence. DMD is a data-driven computational method for detecting and quantifying dynamic events in complex spatiotemporal data. Here, DMD is used to determine photobleaching characteristics of a fluorescent sterol probe, dehydroergosterol (DHE), compared to that of cellular autofluorescence in the nematode Caenorhabditis elegans. It is shown that decomposition of those dynamic modes allows for precise image segmentation, thereby separating probe from autofluorescence without invoking a particular model for the bleaching process. In a second application, DMD of dye-specific photobleaching is used to separate two green-fluorescent dyes, an NBD-tagged sphingolipid and Alexa488-transferrin, thereby assigning them to different cellular compartments. ConclusionsData-based decomposition of dynamic modes can be employed to analyze spatially varying photobleaching of fluorescent probes in cells and tissues for image segmentation, discrimination of probe from autofluorescence and image denoising. The new method should find wide application in analysis of dynamic fluorescence imaging data.

bioinformatics↗

bioRxiv2018

A discontinuous Galerkin model for fluorescence loss in photobleaching of intracellular polyglutamine protein aggregates

BackgroundIntracellular phase separation and aggregation of proteins with extended poly-glutamine (polyQ) stretches are hallmarks of various age-associated neurodegenerative diseases. Progress in our understanding of such processes heavily relies on quantitative fluorescence imaging of suitably tagged proteins. Fluorescence loss in photobleaching (FLIP) is particularly well-suited to study the dynamics of protein aggregation in cellular models of Chorea Huntington and other polyQ diseases, as FLIP gives access to the full spatio-temporal profile of intensity changes in the cell geometry. In contrast to other methods, also dim aggregates become visible during time evolution of fluorescence loss in cellular compartments. However, methods for computational analysis of FLIP data are sparse, and transport models for estimation of transport and diffusion parameters from experimental FLIP sequences are missing.\n\nResultsIn this paper, we present a computational method for analysis of FLIP imaging experiments of intracellular polyglutamine protein aggregates also called inclusion bodies (IBs). By this method, we are able to determine the diffusion constant and nuclear membrane permeability coefficients of polyQ proteins as well as the exchange rates between aggregates and the cytoplasm. Our method is based on a reaction-diffusion multi-compartment model defined on a mesh obtained by segmentation of the cell images from the FLIP sequence. The discontinuous Galerkin (DG) method is used for numerical implementation of our model in FEniCS, which greatly reduces the computing time. The method is applied to representative experimental FLIP sequences, and consistent estimates of all transport parameters are obtained.\n\nConclusionsBy directly estimating the transport parameters from live-cell image sequences using our new computational FLIP approach surprisingly fast exchange dynamics of mutant Huntingtin between cytoplasm and dim IBs could be revealed. This is likely relevant also for other polyQ diseases. Thus, our method allows for quantifying protein dynamics at different stages of the protein aggregation process in cellular models of neurodegeneration.

biophysics↗