Biology subjects

Urbancic, I.

Publications and source records attributed to Urbancic, I..

17 recordsLinked to original sources

bioRxiv2026

Report on pre-validation of an animal-free alternative method (NAM) for regulatory safety testing: InfiniteLungDT, an in-vitro-learned digital twin for the prediction of material-triggered chronic neutrophilic lung inflammation

Until now, there has been no animal-free alternative method for predicting chronic inflammation and delivering the associated dose responses, the timing of onset, and the duration of inflammation, as required by regulatory agencies. We present the results of pre-validation of an in-vitro-learned digital twin (InFiniteLungDT) capable of predicting chronic neutrophilic lung inflammation for regulatory use. The method is based on measuring the dynamics of early biological effects in vitro induced by respirable materials or their mixtures, without the need to know their intrinsic properties. We constructed the digital twin(s) for each of the material, for which we have in vivo exposure data. The instillation data set, comprising 49 different nanomaterials, was used as the primary anchor to calibrate the model. Inhalation data set, comprising 7 different nanomaterials, compliant with OECD TG 412, was used to show the general applicability of the method across species and for different exposure scenaria. In total, about 3094 single mouse exposures and 364 rat exposures (and approx. 775/225 non-exposed mouse/rat controls) were used to predict concentration-dependent time-evolved neutrophil influx into the lung. The accuracy (predictive capacity) of LOAEL determination is 93% for instillation and 84% for inhalation exposure. Taking into account the time-to-deliver-result being less than 1 week, this proves that the effect of inhaled material from acute to chronic conditions can be assessed orders of magnitude faster and cheaper than in a reference animal study.

pharmacology and toxicology↗

bioRxiv2025

HCR-Proxy resolves site-specific proximal RNA proteomes at subcompartmental nanoscale resolution

The spatial organization of RNA condensates is fundamental for understanding of basic cellular functions, but may also provide pivotal insights into diseases. One of the major challenges to understanding the role of condensates is the lack of technologies to map condensate-scale protein architecture at subcompartmental or nanoscale resolution. To address this, we introduce HCR-Proxy, a proximity labelling technique that couples Hybridization Chain Reaction (HCR)-based signal amplification with in situ proximity biotinylation (Proxy), enabling proteomic profiling of RNA-proximal proteomes at subcompartmental resolution. We benchmarked HCR-Proxy using nascent pre-rRNA targets to investigate the distinct proteomic signatures of the nucleolar subcompartments and to uncover a spatial logic of protein partitioning shaped by RNA sequence. Our results demonstrate HCR-Proxys ability to provide spatially-resolved maps of RNA interactomes within the nucleolus, offering new insights into the molecular organisation and compartmentalisation of condensates. This subcompartment-specific nucleolar proteome profiling enabled integration with deep learning frameworks, which effectively confirmed a sequence-encoded basis for protein partitioning across nested condensate subcompartments, characterised by antagonistic gradients in charge, length, and RNA-binding domains. HCR-Proxy thus provides a scalable platform for spatially resolved RNA interactome discovery, bridging transcript localisation with proteomic context in native cellular environments. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/656369v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@116015forg.highwire.dtl.DTLVardef@1b414f1org.highwire.dtl.DTLVardef@1caf0a1org.highwire.dtl.DTLVardef@7bf8b2_HPS_FORMAT_FIGEXP M_FIG C_FIG Bullet points- HCR-Proxy enables the first nanoscale-resolution mapping of RNA-proximal proteomes in situ. - HCR-Proxy establishes a broadly applicable modular platform for spatially resolved RNA- interactomics. - Subcompartmental proteomes are resolved across nucleolar subdomains by targeting specific pre-rRNA regions. - Deep learning confirms a sequence-encoded logic of protein partitioning within condensate subcompartments.

molecular biology↗

bioRxiv2025

Spectral STED microscopy improves spectral sensitivity with polarity-sensitive probes and enables correlative measurements of membrane order and anomalous lipid diffusion

Molecular plasma membrane organization and dynamics play an important role in cellular signalling. Advances in our understanding of the nanoscale architecture of the plasma membrane heavily rely on the development of non-invasive experimental methods, particularly of advanced fluorescence microscopy and spectroscopy techniques with high spatio-temporal resolution and sensitivity to local molecular properties. However, it remains difficult to combine several of them for a multimodal characterisation that would reduce the possibility of misinterpretations. Here, we integrated a spectral detector into a super-resolution stimulated emission depletion (STED) microscope, achieving three goals. First, we show that compared to the standard ratiometric detection using fixed bandpass filters, the spectrally resolved acquisition together with spectral fitting or phasor analysis improves the accuracy of experiments determining membrane lipid order with polarity-sensitive probes multifold. Secondly, we demonstrate that this acquisition scheme allows the use of such probes in combination with other dyes with overlapping spectra, enabling co-localisation of the membrane order maps with other cellular structures of interest, e.g. fluorescently labelled proteins. Finally, we correlate the obtained membrane lipid order with the anomalous trapped diffusion properties of a fluorescent sphingomyelin lipid analogue in the plasma membrane of living cells, as determined by STED fluorescence correlation spectroscopy, and highlight that some of the most apparent trapping sites locate at the boundaries of local ordered environments discernible by the introduced spectral STED microscopy. With additional measurements in model membranes and Monte-Carlo simulations we conclude that for sub-100 nm ordered environments uneven probe partitioning cannot by itself explain the trapping diffusion of SM in cells.

biophysics↗

bioRxiv2024

Integrative analysis of higher-order transcriptome organisation and RNA condensation principles

Complex RNA-protein networks play a pivotal role in the formation of many types of biomolecular condensates. How intrinsic RNA features contribute to condensate formation however remains unclear. Here, we integrate tailored transcriptomics assays to identify a distinct class of developmental condensation-prone RNAs termed smOOPs (semi-extractable, orthogonal organic phase separation-enriched RNAs). These transcripts are localised to larger intracellular foci, form denser RNA-RNA interaction subnetworks than expected and are heavily bound by RNA binding proteins (RBPs). Using an explainable deep learning framework, we reveal that smOOPs harbor characteristic sequence composition with lower sequence complexity, increased intramolecular folding and specific RBP binding patterns. Intriguingly, these RNAs encode proteins bearing extensive intrinsically disordered regions and are markedly predicted to be involved in biomolecular condensates, indicating an interplay between RNA- and protein-based features in phase separation. This work advances our understanding of condensation-prone RNAs and provides a versatile resource to further investigate RNA-driven condensation principles.

molecular biology↗

bioRxiv2024

Particulate matter constituents trigger the formation of extracellular amyloid beta and Tau-containing plaques and neurite shortening in vitro

IntroductionAir pollution is an environmental factor associated with Alzheimers disease, characterized by decreased cognitive abilities and memory. The limited models of sporadic Alzheimers disease fail to replicate all pathological hallmarks of the disease, making it challenging to uncover potential environmental causes. Environmentally driven models of Alzheimers disease are thus timely and necessary. MethodsWe used live-cell confocal fluorescent imaging combined with high-resolution stimulated emission depletion (STED) microscopy to follow the response of neuron-like cells to nanomaterial exposure. Here, we report that a high dose rate in vitro exposure of neuron-like cells to particulate matter constituents reproduces neurodegenerative phenotype, including extracellular amyloid-{beta} containing plaques and decreased neurite length. ResultsConsistent with the existing in vivo research, we observed detrimental effects, specifically a substantial reduction in neurite length and formation of amyloid beta plaques, after exposure to iron oxide and diesel exhaust particles. Conversely, after exposure to engineered cerium oxide nanoparticles, the lengths of neurites were maintained, and almost no extracellular amyloid beta plaques were formed. DiscussionAlthough the exact mechanism behind this effect remains to be explained, the high dose rate in vitro model, comprising wild-type neuron-like cells, could serve as an alternative environmentally driven model of Alzheimers disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/586796v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@63faf1org.highwire.dtl.DTLVardef@1eff3cdorg.highwire.dtl.DTLVardef@6f3365org.highwire.dtl.DTLVardef@973a78_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO High dose rate in vitro exposure of neuron-like cells to particulate matter constituents, like diesel exhaust and iron oxide nanoparticles, reproduces neurodegenerative phenotype, including extracellular amyloid--containing plaques and reduction in neurite length and density. C_FIG

pharmacology and toxicology↗

bioRxiv2024

Binucleated cell formation and oncogene expression after particulate matter exposure is preceded by microtubule disruption, dysregulated cell cycle, prolonged mitosis, and septin binding

Several biopersistent high aspect ratio nanomaterials show pronounced pathogenic effects, from chronic lung inflammation and fibrosis to cancer. For example, asbestos fibers are classified as carcinogens, whereas the carcinogenicity of highly inflammatory multi-wall carbon nanotubes (MWCNTs), and TiO2 nanomaterials is still being evaluated. The exact early mechanisms of their pathogenicity towards inflammation and cancer remains uncertain, but it is likely not due to genotoxic or mutagenic activity. A proposed early mode of action that might lead to the formation of cancerous cells for asbestos fibers is the formation of binucleated and multinucleated cells, resulting in genetic instability. Here, we show that two high aspect ratio nanomaterials, MWCNTs and TiO2 nanotubes, which both induce chronic lung inflammation, induce very different cancer-related changes in vitro. TiO2 nanotubes - but not low aspect-ratio nanocubes of the same crystalline structure - disrupt microtubule organization and prolong mitosis, as well as deform nuclear shape, induce the formation of binucleated cells, and downregulate the tumour suppressor protein p53, whereas only the MWCNTs activate the stimulator of the interferon genes (STING) pathway, a hallmark of lung cancer. The observed differences in cellular responses to different high aspect ratio materials imply the need for assessing each nanomaterial individually with a broad range of tests rather than relying solely on a single marker or pathway, or even morphological or bulk chemical properties to infer possible carcinogenic properties.

cancer biology↗

bioRxiv2023

Quantifying fluorescence lifetime responsiveness of environment sensitive probes for membrane fluidity measurements

The structural diversity of different lipid species within the membrane defines its biophysical properties such as membrane fluidity, phase transition, curvature, charge distribution and tension. Environment-sensitive probes, which change their spectral properties in response to their surrounding milieu, have greatly contributed to our understanding of such biophysical properties. To realize the full potential of these probes and to avoid misinterpretation of their spectral responses, a detailed investigation of their fluorescence characteristics in different environments is necessary. Here, we examined fluorescence lifetime of two newly developed membrane order probes, NR12S and NR12A, in response to alterations in their environments such as degree of lipid saturation, cholesterol content, double bond position and configuration and phospholipid headgroup. As comparison, we investigated lifetime sensitivity of the membrane tension probe Flipper in these environments. Applying fluorescence lifetime imaging microscopy (FLIM) in both model membranes and biological membranes, all probes distinguished membrane phases by lifetime, but exhibited different lifetime sensitivities to varying membrane biophysical properties (e.g. cholesterol). While the lifetime of Flipper is particularly sensitive to membrane cholesterol content, NR12S and NR12A lifetime is moderately sensitive to both cholesterol content and lipid acyl chains. Moreover, all probes exhibit longer lifetimes at longer emission wavelengths in membranes of any complexity. This emission-wavelength dependency results in varying lifetime resolution at different spectral regions, highly relevant for FLIM data acquisition. Our data provides valuable insights on how to perform FLIM with these probes and highlights both their potential and limitations.

biophysics↗

bioRxiv2021

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics. SummaryThe peroxisomal import receptor PEX5 transports newly synthesized proteins from the cytosol to the peroxisomal matrix. Here the cytosolic diffusion and interaction dynamics of PEX5 are characterized by advanced microscopic spectroscopy methods, revealing a so far unknown interaction partner.

biophysics↗

bioRxiv2021

Controlled Fluorescent Labelling of Metal Oxide Nanoparticles for Artefact-free Live Cell Microscopy

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. They are best investigated using non-invasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labelling of nanoparticles with fluorescent dyes. When performed inadequately, unbound fluorophores and inadvertently altered chemical and physical properties of the nanoparticles can, however, result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we here describe a minimal combination of experimental methods to enable artefact-free fluorescent labelling of metal-oxide nanoparticles - the largest subpopulation of nanoparticles by industrial production and applications - and demonstrate its application in the case of TiO2 nanotubes. We 1) characterize potential changes of the nanoparticles surface charge and morphology that might occur during labelling, and 2) assess stable binding of the fluorescent dye to nanomaterial, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials mechanism of action and will foster widespread and safe use of new nanomaterials. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/440400v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@358ffborg.highwire.dtl.DTLVardef@7faaecorg.highwire.dtl.DTLVardef@1ae7fb3org.highwire.dtl.DTLVardef@141c4b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Aggregation and immobilisation of membrane proteins interplay with local lipid order in the plasma membrane of T cells

The quest for understanding of numerous vital membrane-associated cellular processes, such as signalling, has largely focussed on the spatiotemporal orchestration and reorganisation of the identified key proteins, including their binding and aggregation. Despite strong indications of the involvement of membrane lipid heterogeneities, historically often termed lipid rafts, their roles in many processes remain controversial and mechanisms elusive. Taking activation of T lymphocytes as an example, we here investigate membrane properties around the key proteins - in particular the T cell receptor (TCR), its main kinase Lck, and phosphatase CD45. We determine their partitioning and co-localisation in passive cell-derived model membranes (i.e. giant plasma-membrane vesicles, GPMVs), and explore their mobility and local lipid order in live Jurkat T cells using fluorescence correlation spectroscopy and spectral imaging with polarity-sensitive membrane probes. We find that upon aggregation and partial immobilisation, the TCR changes its preference towards more ordered lipid environments, which can in turn passively recruit Lck. We observe similar aggregation-induced local membrane ordering and recruitment of Lck also by CD45, as well as by a membrane protein of antigen-presenting cells, CD86, which is not supposed to interact with Lck directly. This highlights the involvement of lipid-mediated interactions and suggests that the cellular membrane is poised to modulate the frequency of protein encounters according to their aggregation state and alterations of their mobility, e.g. upon ligand binding.

biophysics↗

bioRxiv2020

Towards predictive nanotoxicology: from roundabout of molecular events to chronic inflammation prediction

Nanomaterial-induced diseases cannot be reliably predicted because of the lack of clearly identified causal relationships, in particular between acute exposures and chronic symptoms. By applying advanced microscopies and omics to in vitro and in vivo systems, together with in silico molecular modelling, we have here determined that the long-lasting response to a single exposure originates in the counteracting of a newly discovered nanomaterial quarantining and nanomaterial cycling among different lung cell types. This allows us to predict the nanomaterial-induced spectrum of lung inflammation using only in vitro measurements and in silico modelling. Besides its profound implications for cost-efficient animal-free predictive toxicology, our work also paves the way to a better mechanistic understanding of nanomaterial- induced cancer, fibrosis, and other chronic diseases.

molecular biology↗

bioRxiv2020

Background reduction in STED-FCS using coherent-hybrid STED

Fluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics of living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes at the nanoscale in living cells. In twodimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volumee, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS based investigations of 3D diffusion on the sub-diffraction scale.

biophysics↗

bioRxiv2019

z-STED imaging and spectroscopy to investigate nanoscale membrane structure and dynamics

Super-resolution STED microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy/2D) or axially (z/3D). 2D STED has been extensively used to elucidate the nanoscale membrane structure and dynamics, via imaging or combined with spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and spectral imaging. On the contrary, z-STED has not been used in this context. Here, we show that a combination of z-STED with FCS or spectral imaging enables us to see previously unobservable aspects of cellular membranes. We show that thanks to an axial resolution of approximately 100 nm, z-STED can be used to distinguish axially close-by membranes, early endocytic vesicles or tubular membrane structures. Combination of z-STED with FCS and spectral imaging showed diffusion dynamics and lipid organization in these structures, respectively.

biophysics↗

bioRxiv2019

High photon count rate super-resolution fluorescence fluctuation spectroscopy

Probing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy. Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of up to 40 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01-1 M. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells.

biophysics↗

bioRxiv2019

Mechanical properties of plasma membrane vesicles correlate with lipid order and viscosity and depend on cell density

Plasma membranes dynamically respond to external cues and changing environment. Quantitative measurements of these adaptations can elucidate the mechanism that cells exploit to survive, adapt and function. However, cell-based assays are affected by active processes while measurements on synthetic models suffer from compositional limitations. Here, as a model system we employ giant plasma membrane vesicles (GPMVs), which largely preserve the plasma membrane lipidome and proteome. From analysis of fluorescence emission and lifetime of environment-sensitive dyes, and membrane shape fluctuations, we investigate how plasma membrane order, viscosity and bending rigidity are affected by different stimuli such as cell seeding density in three different cell models. Our studies reveal that bending rigidity of plasma membranes vary with lipid order and microviscosity in a highly correlated fashion. Thus, readouts from polarity- and viscosity-sensitive probes represent a promising indicator of membrane mechanical properties. Quantitative analysis of the data allows for comparison to synthetic lipid membranes as plasma membrane mimetics.

biophysics↗

bioRxiv2019

Adaptive optics allows 3D STED-FCS measurements in the cytoplasm of living cells

Fluorescence correlation spectroscopy in combination with super-resolution stimulated emission depletion microscopy (STED-FCS) is a powerful tool to investigate molecular diffusion with sub-diffraction resolution. It has been of particular use for investigations of two dimensional systems like cell membranes, but has so far seen very limited applications to studies of three-dimensional diffusion. One reason for this is the extreme sensitivity of the axial (3D) STED depletion pattern to optical aberrations. We present here an adaptive optics-based correction method that compensates for these aberrations and allows STED-FCS measurements in the cytoplasm of living cells.

biophysics↗

bioRxiv2018

Lipid composition but not curvature is a determinant of a low molecular mobility within HIV-1 lipid envelope

Human Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from where it buds out. Previous studies have shown that the HIV-1 envelope is a very low mobility environment with the diffusion of incorporated proteins two orders of magnitude slower than in plasma membrane. One of the reasons for this difference is thought to be due to HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing. To further refine the model of the molecular mobility on HIV-1 surface, we here investigated the relative importance of membrane composition and curvature in Large Unilamellar Vesicles of different composition and size (0.2-1 m) by super-resolution STED microscopy-based fluorescence correlation spectroscopy (STED-FCS) analysis. We find that lipid composition and its rigidity but not membrane curvature play an important role in the decreased molecular mobility on vesicle surface thus confirming that this factor is an essential determinant of HIV-1 low surface mobility. Our results provide further insight into the dynamic properties of the surface of individual HIV-1 particles.

biophysics↗