Biology subjects

Muesken, M.

Publications and source records attributed to Muesken, M..

6 recordsLinked to original sources

bioRxiv2026

Arp2/3 complex-dependent actin remodeling is required for efficient RSV uncoating in A549 cells

Productive human respiratory syncytial virus (RSV) cell-entry requires coordinated interactions between viral proteins and host-cell factors at the plasma membrane-actin cortex interface. Branched actin networks remodel this interface, but their precise contribution to the early stages of RSV infection remains unclear. Here, we interfered with Arp2/3 complex-dependent actin filament branching by generating A549 cell lines disrupted for expression of the essential Arp2 subunit by CRISPR/Cas9. Permanent loss of Arp2 reduced the infection of the RSV long GFP reporter virus as quantified over the first 24 h post-infection. Compromised infection efficiency in Arp2 knockout cells persisted at later time points and also resulted in reduced syncytia formation. Notably, these infection phenotypes were not accompanied by obvious changes in viral host cell attachment. Moreover, photoactivated localization microscopy (PALM) studies revealed comparable receptor diffusion and clustering in cells stably expressing mEos3.2-tagged insulin-like growth factor I receptor (IGF1R). Although Arp2/3-deficient cells displayed fewer albeit larger macropinosomes as compared to WT cells, no changes were observed for internalized RSV genome levels. In contrast, Arp2-deficient cells appeared suppressed in viral uncoating efficiency. Consequently, viral mRNA expression and the cellular type III interferon response were reduced. Together, these data reveal that Arp2/3 complex-dependent, branched actin networks contribute to the efficiency of RSV uncoating. ImportanceHuman respiratory syncytial virus (RSV) is a major cause of severe respiratory disease. The infection initiates at the plasma membrane-actin cortex interface, yet the role of actin in productive RSV entry has remained unclear. Using CRISPR/Cas9 disruption of the essential Arp2/3 complex subunit Arp2 in A549 cells, we show that branched actin networks are required for efficient RSV infection. Despite actin network remodeling, photoactivated localization microscopy showed unchanged diffusion and clustering of the RSV receptor IGF1R. Although macropinocytosis was affected in Arp2-deficient cells, RSV attachment and internalization were not influenced. In contrast, a {beta}-lactamase virus-like-particle-based assay revealed a defect in uncoating, followed by reduced viral gene expression and a weaker type III interferon response. These findings define Arp2/3 complex-dependent branched actin networks as a host determinant of RSV uncoating and provide a practical approach to quantify uncoating without engineering the RSV genome.

microbiology↗

bioRxiv2025

Viral infection collapses intracytoplasmic membrane integrity and autotrophic metabolism in ammonia-oxidizing Nitrosomonas europaea

Ammonia-oxidizing bacteria (AOB) catalyze the first and rate-limiting step of nitrification. They are essential for nitrogen cycling in engineered and natural environments, yet little is known about their viruses or the consequences of phage infection for host physiology. Here, we report the isolation and characterization of a novel lytic bacteriophage, vB_NeuP-Nir1 (Nir1), infecting the model AOB Nitrosomonas europaea. Phage Nir1 was highly virulent, ceased ammonia oxidation within hours, and caused complete lysis of host populations even at multiplicities of infection as low as 10-6. Electron microscopy revealed drastic host cell remodeling during infection, including pronounced cell bloating and large-scale disintegration of intracytoplasmic membranes. Integrated transcriptomic and metabolomic analyses showed that loss of these ATP and reducing equivalent generating membrane systems was accompanied by signatures of compromised lipid homeostasis and collapse of autotrophic CO2 fixation. In parallel, Nir1 infection induced metabolic rewiring of the host, including upregulation of uptake systems for nucleic acids, amino acids, and small organic compounds, increased expression of iron acquisition and putative iron-dependent respiratory components, as well as accumulation of metabolites associated with membrane breakdown and stabilization of viral DNA. Together, these results provide the first detailed mechanistic insight into phage-induced host modulation in a chemolithoautotrophic nitrifier. Our study establishes the Nir1-N. europaea system as a model for investigating virus-host interactions in AOB and lays the foundation for assessing the role of phages in shaping nitrification and nitrogen cycling in engineered and natural ecosystems.

microbiology↗

bioRxiv2025

Glial cells promote infection by neurotropic Influenza A viruses in vitro

Although influenza A viruses (IAV) are notorious respiratory pathogens, some IAV strains can reach and replicate in the central nervous system (CNS) in vivo. In particular, highly pathogenic avian influenza viruses (HPAIV) pose a threat for future pandemics and are linked to greater neurotropic potential. Moreover, neurotropic IAV strains have shown a more significant impact on the onset and pathogenesis of neurodegenerative diseases (NDD). However, despite its clinical relevance, the dynamics and cellular tropism of IAV infection in the CNS are not well understood. In this study, we analyzed the replication of HPAIV H7N7 in vitro using a primary murine triple co-culture system comprising neurons, astrocytes, and microglia. We found that microglia become highly infected early on and induce a strong pro-inflammatory response before undergoing apoptosis. Using fluorescence microscopy with automated single-cell profiling, we found that, in contrast to non-neurotropic H3N2, the H7N7 nucleoprotein accumulated in neuronal somata throughout the infection without being transported into dendrites or axons. A combination of single-cell and co-culture replication assays led us to conclude that astrocytes are the primary virus producers of HPAIV H7N7 in the CNS. Our results illuminate the acute phase of neurotropic IAV infection, highlighting its implications for the association between IAV and the development of neurodegenerative diseases.

microbiology↗

bioRxiv2024

Development and Characterization of a 2D Porcine Colonic Organoid Model for Studying Intestinal Physiology and Barrier Function

The porcine colon epithelium plays a crucial role in nutrient absorption, ion transport, and barrier function. However ethical concerns necessitate the development of alternatives to animal models for its study. The objective of this study was to develop and characterise a two-dimensional (2D) in vitro model of porcine colonic organoids that closely mimics native colon tissue, thereby supporting in vitro research in gastrointestinal physiology, pathology, and pharmacology. Porcine colonic crypts were isolated and cultured in three-dimensional (3D) organoid systems, which were subsequently disaggregated to form 2D monolayers on transwell inserts. The integrity of the monolayers was evaluated through the measurement of transepithelial electrical resistance (TEER) and electron microscopy. The functional prerequisites of the model were evaluated through the measurement of the mRNA expression of key ion channels and transporters, using quantitative RT-PCR. Ussing chamber experiments were performed to verify physiological activity. The 2D monolayer displayed robust TEER values and retained structural characteristics, including microvilli and mucus-secreting goblet cells, comparable to those observed in native colon tissue. Gene expression analysis revealed no significant differences between the 2D organoid model and native tissue with regard to critical transporters. Ussing chamber experiments demonstrated physiological responses that were consistent with those observed in native colonic tissue. In conclusion, 2D porcine colonic organoid model can be recommended as an accurate representation of the physiological and functional attributes of the native colon epithelium. This model offers a valuable tool for investigating intestinal barrier properties, ion transport, and the pathophysiology of gastrointestinal diseases, while adhering to the 3R principles.

physiology↗

bioRxiv2023

Isolation and quantification of bacterial membrane vesicles for quantitative metabolic studies using mammalian cell cultures

Bacterial membrane vesicles (BMVs) are produced by most bacteria and participate in various cellular processes, such as intercellular communication, nutrient exchange, and pathogenesis. Notably, these vesicles can contain virulence factors, including toxic proteins, DNA, and RNA. Such factors can contribute to the harmful effects of bacterial pathogens on host cells and tissues. Although the general effects of BMVs on host cellular physiology are well known, the underlying molecular mechanisms are less understood. In this study, we introduce a vesicle quantification method, leveraging the membrane dye FM4-64. We utilize a linear regression model to analyze the fluorescence emitted by stained vesicle membranes to ensure consistent and reproducible vesicle-host interaction studies using cultured cells. This method is particularly valuable for identifying host cellular processes impacted by vesicles and their specific cargo. Moreover, it outcompetes clearly unreliable protein concentration-based methods. We (1) show a linear correlation between the quantity of vesicles and the fluorescence signal emitted from the FM4-64 dye, (2) introduce the "vesicle load" as a new semi-quantitative unit, facilitating more reproducible vesicle-cell culture interaction experiments (3) show that a stable vesicle load yields consistent host responses when studying vesicles from Pseudomonas aeruginosa mutants (4) demonstrate that typical vesicle isolation contaminants, such as flagella, do not significantly skew the metabolic response of lung epithelial cells to P. aeruginosa vesicles, and (5) identify inositol-1-monophosphatase (SuhB) as a pivotal regulator in the vesicle-mediated pathogenesis of P. aeruginosa.

cell biology↗

bioRxiv2021

Lamellipodia-like actin networks in cells lacking WAVE Regulatory Complex

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases mediating activation of WAVE Regulatory Complex (WRC) driving Arp2/3 complex-dependent actin assembly. Previous genome editing studies solidified the view of an essential, linear pathway employing aforementioned components. Using disruption of the WRC subunit Nap1 and its paralogue Hem1 followed by serum and growth factor stimulation or expression of active GTPases now revealed a pathway to formation of Arp2/3 complex-dependent, lamellipodia-like structures (LLS) that require both Rac and Cdc42, but not WRC. These observations were independent of WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from the latter, induced LLS contained all common lamellipodial regulators tested, including cortactin, the Ena/VASP ligand lamellipodin or FMNL subfamily formins. Our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery operating without WRC and Ena/VASP.

cell biology↗