Biology subjects

Blom, H.

Publications and source records attributed to Blom, H..

11 recordsLinked to original sources

bioRxiv2026

Direct visualization of Na,K-ATPase clustering by 3D DNA-PAINT MINFLUX nanoscopy

Direct validation of the nanoscale structural organization of membrane proteins requires localization precision that matches their molecular dimensions. The sodium-potassium pump, or the Na,K-ATPase is an integral membrane protein responsible for maintaining electrochemical gradients and cellular energy homeostasis. Although its crystal structure is characterized, the organization of the Na,K-ATPase within native plasma membranes, particularly whether it forms functional oligomers, remains an open question. Here, we combined 3D MINFLUX nanoscopy with DNA-PAINT with sub-10 nm localization precision to map the clustering topology of the Na,K-ATPase in mammalian cells. By targeting EGFP-tagged Na,K-ATPase 1 and {beta}1 subunits using anti-GFP nanobodies, we obtained high-density 3D localization maps of the protein in the plasma membrane. To evaluate the point patterns, we developed a computational data-driven spatial point assignment approach that segments apical and basal localizations, mitigating clustering artifacts produced by imaging two membranes in close proximity. Furthermore, we used a spatial statistical approach analyzing sequential nearest-neighbour distances to elucidate supramolecular arrangement information. Our data reveal a preferential nearest-neighbour distance of approximately 7 nm, providing direct visual confirmation of Na,K-ATPase dimerization. Additionally, we identified higher-order nanoclusters composed of up to 21 proteins. These findings provide definitive structural evidence of the dimeric configuration of Na,K-ATPase, establishing a foundation for future research on the functional and regulatory implications of Na,K-ATPase clustering.

biophysics↗

bioRxiv2025

Dual-color expansion microscopy of membrane proteins using bioorthogonal labelling

Site-specific incorporation of non-canonical amino acids (ncAAs) combined with bioorthogonal click chemistry provides a powerful tool for fluorescent protein labeling, overcoming the linkage error inherent to antibody-based probes. In this study, we present the development of dual-color super-resolution imaging utilizing ncAA labeling together with expansion microscopy (ExM). After optimizing the labeling procedures and fluorophore selection, we visualize and resolve the nanoscale distribution of Na,K-ATPase 1 and {beta}1 subunits in expanded HEK 293T cells. We validate our approach by super-resolution STED imaging of ncAA labeled {beta}1 subunit in unexpanded cells. This work establishes a robust framework for multiplexed, high-resolution imaging and suggests that the combination of ncAA labeling with ExM has the potential to push biological imaging toward [A]ngstrom-level resolution.

biophysics↗

bioRxiv2025

Spatial mapping of DNA synthesis reveals dynamics and geometry of human replication nanostructures

DNA replication is essential to life and ensures the accurate transmission of genetic information, which is significantly disturbed during cancer development and chemotherapy. While DNA replication is tightly controlled in time and space, methods to visualise and quantify replication dynamics within 3D human cells are lacking. Here, we introduce 3D-Spatial Assay for Replication Kinetics (3D-SPARK), an approach enabling nanoscale analysis of DNA synthesis dynamics in situ. 3D-SPARK integrates optimised nucleotide analogue pulse labelling with super-resolution microscopy to detect, classify, and quantify replication nanostructures in single cells. By combining immunofluorescence techniques with click chemistry-based nascent DNA labelling and transfection of fluorescent nucleotide derivatives, we map multi-colour DNA synthesis events in relation to established replication proteins, local RNA-protein condensates or large subnuclear domains. We demonstrate quantitative changes in size, relative abundance and spatial arrangement of nanoscale DNA synthesis events upon chemotherapeutic treatment, CDC6 oncogene expression and loss of chromatin organiser RIF1. The flexibility, precision and modular design of 3D-SPARK helps bridging the gap between spatial cell biology, genomics, and 2D fibre-based replication studies in health and disease.

cell biology↗

bioRxiv2024

Revised diffusion law permits quantitative nanoscale characterization of membrane organization

Formation of functional nanoscopic domains is an inherent property of plasma membranes. Stimulated emission depletion combined with fluorescence correlation spectroscopy (STED-FCS) has been used to identify such domains, however, the information obtained by STED-FCS has been limited to presence of such domains while crucial parameters have not been accessible, such as size (Rd), the fraction of occupied membrane surface (f), in-membrane lipid diffusion inside (Din) and outside (Dout) the nanodomains as well as their self-diffusion (Dd). Here, based on a revision of the diffusion law, we present an approach to retrieve these five parameters from STED-FCS data. We verify that approach on ganglioside nanodomains in giant unilamellar vesicles (GUVs), validating the Saffman-Delbruck assumption for Dd. We examined STED-FCS data in both plasma membranes of living PtK2 cells and in giant plasma membrane vesicles (GPMVs) and present a quantitative framework for molecular diffusion modes in biological membranes.

biophysics↗

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↗

bioRxiv2023

Mechanical forces at the kidney filtration barrier govern spatial orientation of podocyte processes on capillaries

Mammalian kidneys filter enormous volumes of water and small solutes, a filtration driven by the very high hydrostatic pressure in glomerular capillaries. Interdigitating cellular processes of podocytes form the slits for fluid filtration. They are connected by the membrane-like slit diaphragm cell junction containing a mechanosensitive ion channel complex and allow filtration while counteracting hydrostatic pressure. Using high-resolution microscopy, we show that filtration-slit-generating secondary processes preferentially align along the capillaries longitudinal axis while primary processes are preferably perpendicular to the longitudinal direction. The preferential orientation requires maturation in development and is lost in disease states. We demonstrate that loss of proper orientation might contribute to impaired filtration by collapsing of the filtration slits and reducing the mechanical stability of podocyte processes. Together, these data suggest that podocytes sense mechanical strain to utilize circumferential hoop stress balancing the massive mechanical strain generated from fluid flow over the filtration slit.

cell biology↗

bioRxiv2021

Three-dimensional Super-resolved Imaging of Paraffin-embedded Kidney Samples

Diseases of the glomeruli, the renal filtration units, are a leading cause of progressive kidney disease. Assessment of the ultrastructure of podocytes at the glomerular filtration barrier is essential for diagnosing diverse disease entities, providing insight into the disease pathogenesis as well as monitoring treatment responses. New technologies, including superresolved nanoscopy and expansion microscopy, as well as new sample preparation techniques, are starting to revolutionize imaging of biopsy specimens. However, our previous approaches for simple and fast three-dimensional imaging of optically cleared samples are to date not compatible with formalin fixed paraffin-embedded (FFPE) tissue, impeding application in clinical routine. Here we provide protocols that circumvent these limitations and allow for three dimensional STED and confocal imaging of FFPE kidney tissue with similar staining and image quality as compared to our previous approaches. This would increase the feasibility to implement these protocols in clinical routines, as FFPE is the gold standard method for storage of patient samples.

pathology↗

bioRxiv2021

Deep learning-based segmentation and quantification of podocyte foot process morphology

The kidneys constantly filter enormous amounts of fluid, with almost complete retention of albumin and other macromolecules in the plasma. Diseases of podocytes at the kidney filtration barrier reduce the intrinsic permeability of the capillary wall resulting in albuminuria. However, direct quantitative assessment of the underlying morphological changes has previously not been possible. Here we developed a deep learning-based approach for segmentation of foot processes in images acquired with optical microscopy. Our method - Automatic Morphological Analysis of Podocytes (AMAP) - accurately segments foot processes and robustly quantifies their morphology. It also robustly determined morphometric parameters, at a Pearson correlation of r > 0.71 with a previously published semi-automated approach, across a large set of mouse tissue samples. The artificial intelligence algorithm wasWe applied the analysis to a set of human kidney disease conditions allowing comprehensive quantification of various underlying morphometric parameters. These results confirmed that when podocytes are injured, they take on a more simplified architecture and the slit diaphragm length is significantly shortened, resulting in a reduction in the filtration slit area and a loss of the buttress force of podocytes which increases the permeability of the glomerular basement membrane to albumin.

pathology↗

bioRxiv2021

The extinction and survival of sharks across the end-Cretaceous mass extinction

Sharks (Selachimorpha) are iconic marine predators that have survived multiple mass extinctions over geologic time. Their fossil record is represented by an abundance of teeth, which traditionally formed the basis for reconstructing large-scale diversity changes among different selachimorph clades. By contrast, corresponding patterns in shark ecology, as measured through morphological disparity, have received comparatively limited analytical attention. Here, we use a geometric morphometric approach to comprehensively examine the dental morphology of multiple shark lineages traversing the catastrophic end-Cretaceous mass extinction -- this event terminated the Mesozoic Era 66 million years ago. Our results show that selachimorphs maintained virtually static levels of dental disparity in most of their constituent clades during the Cretaceous/Paleogene transition. Nevertheless, selective extinctions did impact on apex predator lineages characterized by triangular blade-like teeth, and in particular, lamniforms including the dominant Cretaceous anacoracids. Other groups, such as, triakid carcharhiniforms, squalids, and hexanchids, were seemingly unaffected. Finally, while some lamniform lineages experienced morphological depletion, others underwent a post-extinction disparity increase, especially odontaspidids, which are typified by narrow-cusped teeth adapted for feeding on fishes. This disparity shift coincides with the early Paleogene radiation of teleosts, a possible prey source, as well as the geographic relocation of shark disparity hotspots, perhaps indicating a regionally disjunct pattern of extinction recovery. Ultimately, our study reveals a complex morphological response to the end-Cretaceous mass extinction event, the dynamics of which we are only just beginning to understand.

paleontology↗

bioRxiv2020

Dental ontogeny in the most primitive bony fish Lophosteus reveals the developmental relationship between teeth and dermal odontodes

Ontogenetic data obtained by synchrotron microtomography of a marginal jawbone of Lophosteus superbus (Late Silurian, 422 Million years old), the phylogenetically basalmost stem osteichthyan, reveal developmental relationships between teeth and ornament that are not obvious from the adult morphology. The earliest odontodes are two longitudinal founder ridges formed at the ossification center. Subsequent odontodes that are added lingually to the ridges turn into conical teeth and undergo cyclic replacement, while those added labially achieve a stellate appearance. The stellate odontodes deposited directly on the bony plate are aligned with the alternate files of the teeth. Successive odontodes overgrowing the labial tooth rows become tooth-like and the replacement teeth near to them are ornament-like. We propose that teeth and ornament are modifications of a single odontode system regulated and differentiated by the oral and dermal signals; signal cross-communication between the two domains can occur around the oral-dermal boundary.

evolutionary biology↗

bioRxiv2020

Screening for inborn errors of metabolism using untargeted metabolomics and out-of-batch controls

MotivationUntargeted metabolomics is an emerging technology in the laboratory diagnosis of inborn errors of metabolism (IEM). In order to judge if metabolite levels are abnormal, analysis of a large number of reference samples is crucial to correct for variations in metabolite concentrations resulting from factors such as diet, age and gender. However, a large number of controls requires the use of out-of-batch controls, which is hampered by the semi-quantitative nature of untargeted metabolomics data, i.e. technical variations between batches. Methods to merge and accurately normalize data from multiple batches are urgently needed. Methods & resultsBased on six metrics, we compared existing normalization methods on their ability to reduce batch effects from eight independently processed batches. Many of those showed marginal performances, which motivated us to develop Metchalizer, a normalization method which uses 17 stable isotope-labeled internal standards and a mixed effect model. In addition, we propose a regression model with age- and sex as covariates fitted on control samples obtained from all eight batches. Metchalizer applied on log-transformed data showed the most promising performance on batch effect removal as well as in the detection of 178 known biomarkers across 45 IEM patient samples and performed at least similar to an approach using 15 within-batch controls. Furthermore, our regression model indicates that 10-24% of the considered features showed significant age-dependent variations. ConclusionsOur comprehensive comparison of normalization methods showed that our Log-Metchalizer approach enables the use out-of-batch controls to establish clinically-relevant reference values for metabolite concentrations. These findings opens possibilities to use large scale out-of-batch control samples in a clinical setting, increasing throughput and detection accuracy. AvailabilityMetchalizer is available at https://github.com/mbongaerts/Metchalizer/

bioinformatics↗