Biology subjects

Weissinger, H.

Publications and source records attributed to Weissinger, H..

3 recordsLinked to original sources

bioRxiv2026

Recipient cell identity governs intracellular transport and membrane interactions of extracellular vesicles across species

Extracellular vesicles (EVs) function as a natural communication system, enabling transfer of biomolecules between cells. Although EVs are produced by nearly all cell types and have shown promise as therapeutic agents, our understanding of how EVs from different cellular origins recognize recipient cells, are taken up, and processed intracellularly remains limited. In particular, it remains unclear whether intracellular EV behavior is primarily governed by EV-intrinsic properties or by the recipient-cell environment. Here, we systematically investigate the relative contributions of EV origin and recipient-cell identity to intracellular EV trafficking. By combining single-vesicle tracking with machine learning-based diffusion-state classification, we analyzed EVs derived from four distinct sources across three recipient cell types over three different time spans. This approach enabled characterization of dynamic transport behaviors, overcoming limitations of traditional ensemble-averaging methods. We observed recipient cell-dependent differences in intracellular trafficking patterns, ranging from relatively uniform and dynamic motion to more heterogeneous and confined behaviors, reflecting variability in intracellular transport environments. Despite these differences, EV origin had only a modest influence on intracellular dynamics following uptake. These findings suggest a model in which intracellular transport arises from an interplay between general EV-associated properties and cell-specific environment.

biophysics↗

bioRxiv2026

Unmasking the diversity of extracellular nucleic acids in the biofilm matrix using nucleic acid-binding dyes

Extracellular nucleic acids (eNA) are central components of bacterial biofilms, contributing to structural integrity, antibiotic tolerance, and emerging functions such as extracellular electron transfer and peroxidase-like catalysis. While extracellular DNA has traditionally been assumed to adopt the canonical B-DNA conformation, biofilms are now known to contain non-canonical structures, including Z-DNA/RNA (Z-NA), G-quadruplex DNA/RNA (G4-NA), and substantial amounts of extracellular RNA. Conventional nucleic acid-binding dyes are widely used for rapid eNA detection, yet their specificity for these diverse structures has not been systematically evaluated. Here, we compare the fluorescence properties of eleven membrane-impermeant dyes (TOTO, BOBO, YOYO, and POPO series, SYTOX Green, SYTOX Red, and propidium iodide) against synthetic B-DNA, Z-DNA, G4-DNA, A-RNA, Z-RNA, and G4-RNA oligonucleotides, with Z-NA stabilised through brominated guanosine analogues synthesised in-house. A clear pattern emerged: green-fluorescent dyes preferentially bound canonical B-DNA, whereas red-fluorescent counterparts displayed broader specificity that extended to non-canonical structures. TOTO-3 and SYTOX Red bound G4-NA with higher fluorescence than B-DNA, and propidium iodide showed an unexpected preference for A-RNA over B-DNA. These observations were validated in Staphylococcus aureus biofilms by parallel immunolabelling with structure-specific antibodies. TOTO-3, YOYO-3, BOBO-3, POPO-3, and propidium iodide reproduced the eNA distribution at the bacterial cell surface. Finally, we introduce poly-A tailing with fluorescently labelled ATP as a stringent, RNA-specific imaging method for biofilms. Together, these results provide practical guidelines for visualising the structural diversity of eNA in biofilms. HIGHLIGHTS- Biofilms contain non-canonical structures of extracellular DNA and RNA - This study tests the ability of DNA-binding dyes to visualise such structures - Propidium iodide visualises RNA with brighter fluorescence than DNA - Red-fluorescent dyes were more versatile than green-fluorescent dyes - Combining several dyes enabled the detection of non-canonical structures

microbiology↗

bioRxiv2025

Native extracellular vesicles display surface bound RNAs that are co-delivered to cells

Extracellular vesicles (EVs) can transport functional RNA between cells and therefore hold great potential for diagnostics and RNA-based therapeutics. Classically, RNA is believed to be encapsulated in the EV lumen. However, it has recently been demonstrated that cells present RNA on their surface. This RNA was found to be glycosylated, and although glycosylated tRNA was also found in EVs, its exact location remained elusive. Here, we demonstrate the presence of RNA on the surface of mesenchymal stem cell (MSC) derived EVs. By combining single-vesicle measurements with direct and selective visualization of RNA, we introduce surface RNA (surfRNA) as a new inherent component of EVs. RNA sequencing supports the surface localization of this RNA and further identifies tRNA fragments as primary constituent of surfRNA. Importantly, surfRNA is co-delivered to target cells together with EVs, suggesting a yet unrecognized uptake route of extracellular RNA. A deeper understanding of the surface-associated RNA may have significant implications for EV biogenesis, targeting, and downstream functional effects. We further envision that these findings are transferable to other nanoparticles and will thereby advance the field of therapeutic RNA delivery.

molecular biology↗