Biology subjects

Weinhardt, V.

Publications and source records attributed to Weinhardt, V..

9 recordsLinked to original sources

bioRxiv2025

Decoding acute neuroinflammatory states from the 3D architecture of in vitro microglia

Microglia, core brain immune defenders, rapidly polarize into pro- or anti-inflammatory states that shape neuronal survival during acute brain inflammation. Yet, how these inflammatory states are encoded at the native cellular level remains unclear. While microglial states associate with specific molecular and organelle markers, it is unknown whether their cellular architecture integrates robust metabolic and structural features. Here, we quantitatively decode inflammatory states from the 3D architecture of individual in vitro mouse microglia (BV-2). Using soft X-ray tomography on established BV-2 polarization, we identify coordinated intracellular organization distinguishing homeostatic, pro-inflammatory, and anti-inflammatory cells, including characteristic lipid droplet-endosome architectures. We further link lipid-endosome reorganization with mTORC1 pathway, by functionally implicating sestrin-2 in promoting anti-inflammation. Finally, we resolve the time-dependent remodeling of lipid droplet 3D profiles by their distinct lipidomic composition across inflammatory states. Overall, our work enables deciphering microglia states in disease-relevant models, with potential to ultimately understand brain immunity.

cell biology↗

bioRxiv2025

Extrinsic polarity cues control lamination versus cluster-based organisation in vertebrate retinal development

1.Photosensitive organs are essential for most animals to perceive and respond to their environment. While the gene regulatory networks establishing retinal identity are deeply conserved across metazoans (reviewed in Gehring, 2012; Vopalensky & Kozmik, 2009; Hahn et al., 2023), the retinal architecture varies widely--from invertebrate compound eyes to vertebrate camera-type eyes (Lamb et al., 2007; Schwab, 2017; Arendt & Wittbrodt, 2001). Despite this morphological diversity, early eye anlagen in both, invertebrates and vertebrates, share an initial pseudo-stratified epithelial organization (Weasner & Kumar, 2022; Randlett et al., 2010; Das et al., 2003; Kitambi & Malicki, 2008), which is maintained and elaborated into multi-layered retinae in vertebrates. In contrast, the invertebrate neuroepithelium is re-organized as ommatidia develop. Laminar organisation of the vertebrate retina appears to be a consequence of initial polarisation of the retinal neuroepithelium. This is, however, challenging to test in the organismal context. To address the plasticity of retinal architecture and the impact of epithelial polarity on the structuring of retinal tissue, we take advantage of retinal organoids derived from medaka (Oryzias latipes) (Zilova et al., 2021) that allow to modulate polarity cues and test their impact on the level of epithelialization and structural organisation of the forming retina. We show that under specific culture conditions, medaka retinal organoids undergo a striking morphological switch depending on the level of apico-basal polarity imposed. When polarity cues are continuously provided, a laminated retinal epithelium is established in the organoid. The absence of polarity cues results in the formation of horizontal cellular clusters containing the retinal cell types, which form the vertical retinal column in the developing embryo. We demonstrate that the emergence of this alternative retinal architecture is associated with a loss of epithelial polarity, notably the absence of extracellular matrix (ECM) components, such as laminin, which efficiently rescues lamination. Our findings indicate that tissue-level polarization and lamination in vertebrate retinae require specific extrinsic cues, and that in their absence, differentiating retinal cell types self-organize into structurally distinct, retinal units. This reveals an unexpected plasticity in vertebrate retinal development and indicates a potential for alternative modes of retinal patterning. O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/688026v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@100bb1eorg.highwire.dtl.DTLVardef@fceb9borg.highwire.dtl.DTLVardef@d036a7org.highwire.dtl.DTLVardef@1b41483_HPS_FORMAT_FIGEXP M_FIG C_FIG Retinal cells in medaka organoids adopt either a continuous layered epithelium when supported by laminin or a unit-based, ommatidia-like organization when epithelial continuity is lost. This dual outcome suggests that epithelial integrity represents a branching point between vertebrate and invertebrate strategies of retinal patterning, providing an experimental system to replay alternative evolutionary trajectories of eye design.

developmental biology↗

bioRxiv2025

Interior soft x-ray tomography with sparse global sampling

Objective. To investigate the feasibility of interior imaging reconstruction in soft X-ray tomography to achieve higher spatial resolution cellular imaging, including whole-cell imaging. Approach. We develop an alignment and reconstruction algorithm that enables a combination of a low number of images from sparse whole-cell imaging with a high-resolution local interior scan. Based on numerical simulations, we demonstrate that combined reconstructions mitigate the depth of field limitation in high-resolution scans, enable radiation dose optimization, and yield quantitative X-ray absorption values with sparse sampling. Furthermore, we validate our numerical approach using experimental data from two different cell types and demonstrate that combined reconstruction is a reliable method for obtaining high and local spatial resolution within the volume of a whole cell. Significance. The developed sparse reconstruction algorithm provides a robust and faithful visualization of cellular organelles with soft X-ray tomography. A mesoscale imaging approach, such as an interior tomography scan, enables one to "scout" and zoom into the volumes of interest that contain organelles of interest. Utilizing sparse reconstructions, this increase in spatial resolution is achieved without sacrificing larger volume imaging, providing information on the relative position of all organelles within a cell.

cell biology↗

bioRxiv2024

Soft X-ray tomography reveals variations in B.subtilis biofilm structure upon tasA deletion

Bacterial biofilms are complex communities of cells within a self-produced extracellular matrix. They play crucial roles in healthcare, nutrition, agriculture and environmental research, yet an analysis of their elaborate 3D architecture remains challenging. Understanding mechanisms of biofilm formation, particularly the effects of chemical, physical, and genetic influences or modifications, is crucial but requires structural information at subcellular resolution to enable a community-level analysis of biofilms. In this work, we developed a "biofilm-in-capillary" growth method compatible with full-rotation soft X-ray tomography, providing high-resolution 3D imaging of bacterial cells and their surrounding extracellular matrix during biofilm formation, without drying or fixation steps. This approach offers 50 nm isotropic spatial resolution, rapid imaging time, and quantitative native analysis of biofilm structure. We demonstrate the potential of our method using Bacillus subtilis biofilms, detecting coherent alignment and chaining of wild-type cells while they are travelling towards the oxygen-rich capillary tip region. In stark contrast, the genetic knock-out {Delta}tasA shows a loss of cellular orientation, including changes in the extracellular matrix in volume and chemical density. Notably, we show that the addition of TasA protein to a culture of a {Delta}tasA strain restores the extracellular matrix density and leads to a compaction of cell assemblies, yet no chaining is observed as for the wildtype. Our approach to imaging biofilms is scalable and transferable, opening new avenues for examining biofilm structure and function across various species, including mixed biofilms, and observing 3D reorganization in response to genetic and environmental factors.

microbiology↗

bioRxiv2023

Visualisation of gene expression within the context of tissues: an X-ray computed tomography-based multimodal approach

The development of an organism is orchestrated by the spatial and temporal expression of genes. Accurate visualisation of gene expression patterns in the context of the surrounding tissues offers a glimpse into the mechanisms that drive morphogenesis. We developed correlative light-sheet fluorescence microscopy and X-ray computed tomography approach to map gene expression patterns to the whole organisms 3D anatomy at cellular resolution. We show that this multimodal approach is applicable to gene expression visualised by protein-specific antibodies and fluorescence RNA in situ hybridisation, offering a detailed understanding of individual phenotypic variations in model organisms. Furthermore, the approach provides a unique possibility to identify tissues together with their 3D cellular and molecular composition in anatomically less-defined in vitro models, such as organoids. We anticipate that the visual and quantitative insights into the 3D distribution of gene expression within tissue architecture, by the multimodal approach developed here, will be equally valuable for reference atlases of model organisms development, as well as for comprehensive screens and morphogenesis studies of in vitro models.

developmental biology↗

bioRxiv2022

Extending of imaging volume in soft x-ray tomography

Soft x-ray tomography offers rapid whole single cell imaging with a few tens of nanometers spatial resolution without fixation or labelling. At the moment, this technique is limited to 10 {micro}m thick specimens, such that applications of soft x-ray tomography to large human cells or multicellular specimens are not possible. We have developed a theoretical and experimental framework for soft x-ray tomography to enable extension of imaging volume to 18 {micro}m thick specimens. This approach, based on long depth of field and half-acquisition tomography, is easily applicable to existing full-rotation based microscopes. This opens applications for imaging of large human cells, which are often observed in cancer research and cell to cell interactions.

cell biology↗

bioRxiv2021

Fish primary embryonic stem cells self-assemble into retinal tissue mirroring in vivo early eye development

Organoids derived from pluripotent stem cells promise the solution to current challenges in basic and biomedical research. Further progress and widespread applications are however limited by long developmental time, variable success, and lack of direct comparison to an in vivo reference. To overcome those limitations, we derived organoids from rapidly developing teleosts. We demonstrate how primary embryonic stem cells from zebrafish and medaka efficiently self-organize into anterior neural structures, particularly retina. Within four days, blastula-stage cell aggregates reproducibly execute key steps of eye development: retinal specification, morphogenesis and differentiation. The number of aggregated cells as well as genetic factors crucially impacted upon the concomitant morphological changes that were intriguingly reflecting the in vivo situation. High reproducibility and rapid development of fish-derived organoids in combination with advanced genome editing techniques immediately allow addressing aspects of development and disease, and systematically probing the impact of the physical environment on morphogenesis and differentiation.

developmental biology↗

bioRxiv2018

Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties

Collective phase transitions, including phase separation and gelation of multivalent protein and RNA molecules appears to underlie the biogenesis of biomolecular condensates such as membraneless organelles. In vivo, these condensates encompass hundreds of distinct types of molecules that are often organized into multi-layered structures supporting the differential partitioning of molecules into distinct regions with distinct material properties. The interplay between driven (active) versus spontaneous (passive) processes that are required for enabling the formation of condensates with coexisting layers of distinct material properties remains unclear. Here, we investigate the role of spontaneous driving forces as determinants of protein-RNA condensates with complex morphologies and distinct material properties. Through the use of systematic in vitro experiments and simulations based on coarse-grained models we find that that the collective interactions among the simplest, biologically relevant proteins and archetypal RNA molecules are sufficient for driving the spontaneous emergence of multi-layered condensates with distinct material properties. Our results demonstrate that key properties of protein-RNA condensates such as their overall morphologies, internal dynamics, and the selective partitioning of substrates are governed specific amino acid chemistries as well as RNA sequence and secondary structure. Our findings yield a clear set of heuristics regarding homo- and heterotypic interactions that are likely to be relevant for understanding the interplay between active and passive processes that control the formation of functional biomolecular condensates.

biophysics↗

bioRxiv2018

PSF correction in soft x-ray tomography

In this manuscript, we introduce a linear approximation of the forward model of soft x-ray tomography (SXT), such that the reconstruction is solvable by standard iterative schemes. This linear model takes into account the three-dimensional point spread function (PSF) of the optical system, which consequently enhances the reconstruction data. The feasibility of the model is demonstrated on both simulated and experimental data, based on theoretically estimated and experimentally measured PSFs.

cell biology↗