Biology subjects

Sieben, C.

Publications and source records attributed to Sieben, C..

16 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↗

bioRxiv2026

Label-free detection of individual virus-infected cells using deep learning

Numerous applications in research and medicine rely on reliable identification and quantification of virus-infected cells. Current methods either apply reporter viruses, that often differ from clinical isolates (e. g. cell tropism, immune evasion) or staining approaches, that prevent live-cell experiments and may introduce biases through manual counting. We present a deep learning model for the label-free identification of virus-infected cells on light microscopy images (VAIruScope). To overcome limitations, our pipeline enables an automated quantification of virus-infected cells based on the recognition of cytopathic effects. The method was applied to different cell models and four clinically relevant prototype viruses representing RNA- (influenza A virus), DNA- (human cytomegalovirus, herpes simplex virus-1) and retroviruses (human immunodeficiency virus-1). VAIruScope identified infected cells achieving classification accuracies of up to 96 %. As proof-of-concept, the method was validated using electron microscopy for a wild-type HSV-1. VAIruScope may be applicable to live-cell imaging to investigate infection dynamics.

bioinformatics↗

bioRxiv2025

Molecular insights into the capsular polysaccharide transporter Wza-Wzc complex

Capsular polysaccharides (CPS), which form the protective outer capsule surrounding many Gram-negative bacterial pathogens, are critical virulence determinants. Their biosynthesis is primarily carried out via the conserved Wzx/Wzy-dependent pathway. In Escherichia coli, Group 1 CPS transport through the bacterial envelope is thought to be mediated by the Wza-Wzc complex. In this study, we present the first structural characterization of the complete Wza-Wzc complex from E. coli K12, determined using single-particle cryogenic electron microscopy. The structure revealed an elongated, continuous channel spanning the entire envelope, which is crucial for efficient CPS secretion, as supported by mutagenesis studies. Multiple structural snapshots of the ADP-bound Wza-Wzc complex captured intermediate conformations of the double membrane assembly, highlighting its remarkable intrinsic dynamics. In-depth analysis of the isolated Wza translocon and Wzc co-polymerase, revealed new mechanistic details of both complex formation and CPS transport. Importantly, we identified the jellyroll domain of Wzc as a previously unrecognized CPS-binding module, likely guiding CPS repeat units into a proposed Wzc-Wzy polymerization platform. Collectively, these findings provide new structural and functional insights into CPS synthesis and transport, advancing our understanding of bacterial capsule formation and virulence mechanisms.

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

Ninjurin-1 mediated plasma membrane rupture is a two-step process requiring cell swelling

Ninjurin-1 (NINJ1) executes cell lysis by polymerizing into filaments, yet how these form membrane lesions is unknown. Integrating cell-based assays with molecular dynamics simulations, we demonstrate that NINJ1-driven membrane rupture starts with the lateral association of NINJ1 dimers into double filaments. Subsequently, swelling of necrotic cells increases plasma membrane tension, leading to separation of the filament interface and NINJ1 lesion opening. Inhibiting cell swelling prevents this increase in tension, blocking NINJ1-mediated plasma membrane rupture without disrupting NINJ1 oligomerization, implicating membrane tension as key for opening NINJ1 lesions. The NINJ1 homolog NINJ2 oligomerizes but fails to form membrane lesions during cell death due to the greater stability of its dimer. Finally, using atomic force microscopy of NINJ1-containing proteoliposomes, we show that NINJ1 forms large pores by stabilizing membrane edges. In summary, our findings support a model whereby membrane tension drives a zipper-like opening of NINJ1 double filaments, initiating necrotic cell lysis.

immunology↗

bioRxiv2024

Single influenza A viruses induce nanoscale cellular reprogramming at the virus-cell interface

Viruses, as nanoscale entities with limited proteomes, must efficiently infect target cells using their available resources. During infection, individual virions induce specific cellular signaling within the virus-cell interface, a nanoscale patch of the plasma membrane in contact with the virus. However, virus-induced receptor recruitment and cellular activation are transient processes that occur within minutes and at the nanoscale level. Hence, the temporal and spatial kinetics of such early events often remain poorly understood due to technical limitations. To address this challenge, we developed a novel protocol to covalently immobilize unmodified influenza A viruses on glass surfaces before exposing them to live epithelial cells. This approach extends the observation time for virus-plasma membrane interaction while preserving the viruses native state for uncompromised cell interaction. Using single-molecule super-resolution microscopy, we investigated virus-receptor interaction showing that viral receptors are not immobilized by the virus but rather slowed down, which leads to a specific local receptor accumulation and turnover. We further followed the dynamics of clathrin-mediated endocytosis at the single-virus level and demonstrate the recruitment of adaptor protein 2 (AP-2), previously thought to be uninvolved in influenza A virus infection. Finally, we examined the nanoscale organization of the actin cytoskeleton at the virus-binding site, showing a local and dynamic response of the cellular actin cortex to the infecting virus. Our findings provide novel insights into the fundamental process of virus-cell interaction and demonstrate the versatility and potential impact of our approach on virus-cell biology.

microbiology↗

bioRxiv2024

The bat Influenza A virus subtype H18N11 induces nanoscale MHCII clustering upon host cell attachment

Prior to the discovery of the bat influenza A virus (IAV) subtypes H17N10 and H18N11, it was believed that all IAVs bind via the viral hemagglutinin (HA) to sialic acid residues to mediate attachment and subsequent viral entry. Both HA subtypes, H17 and H18, however, do not bind to sialic acids but instead engage a proteinaceous receptor: major histocompatibility complex class II (MHCII). The mechanistic details of this hitherto unknown protein-mediated entry are not understood. Since conventional IAVs require attachment to clusters of sialylated glycans to overcome the low affinity of the HA-sialic acid interaction, we hypothesized that bat HA would likewise interact with multiple MHCII molecules. Here, we used photoactivated localization microscopy (PALM) on fixed and live cells expressing MHCII fused to appropriate fluorescent reporters. We show that bat IAV particles attach to pre-existing MHCII clusters present on susceptible cells and that the local HA-MHCII interaction results in an increased cluster size. To measure the impact of viral attachment on the dynamics of MHCII, we utilized an experimental setup designated "inverse infection" where intact viral particles were immobilized on coverslips before live MHCII-expressing cells were seeded on top. Recording the trajectories of single MHCII molecules, this approach revealed that the mobility of MHCII was indeed slowed down in viral proximity leading to a local enrichment of MHCII molecules beneath the viral particle. Taken together, these data suggest that attachment of viral particles leads to clustering of MHCII, a process similar to the MHCII dynamics observed during the formation of an immunological synapse.

microbiology↗

bioRxiv2024

An unsupervised deep learning framework encodes super-resolved image features to decode bacterial cell cycle

Super-resolution microscopy can resolve cellular features at the nanoscale. However, increased spatial resolution comes with increased phototoxicity, and reduced temporal resolution. As a result, studies that require the highest spatial resolutions often rely on static or fixed images, lacking dynamic information. This is particularly true of bacteria, whose lateral dimensions approach the scale of the diffraction limit. In this work, we present Enso, a method based on unsupervised machine learning to recover bacterial cell cycle and cell type information from static single molecule localization microscopy (SMLM) images, whilst retaining nanoscale spatial resolution. Enso uses single-cell images as input, and orders cells according to their spatial pattern progression, ultimately linked to the cell cycle. Our method requires no a priori knowledge or categories, and is validated on both simulated and user-annotated experimental data.

bioinformatics↗

bioRxiv2023

An improved workflow for the quantification of orthohantavirus infection using automated imaging and flow cytometry

Determination of the infectious titer is a central requirement when working with pathogenic viruses. The plaque or focus assay is commonly used but a labor- and time-consuming approach to determine the infectious titer of orthohantavirus samples. We have developed an optimized virus quantification approach that relies on the fluorescence-based detection of the orthohantavirus nucleocapsid protein (N) in infected cells with high sensitivity. We present the use of flow cytometry but highlight fluorescence microscopy in combination with automated data analysis as an attractive alternative to increase the information retrieved from an infection experiment. Additionally, we offer an open-source software equipped with a user-friendly graphical interface, eliminating the necessity for advanced programming skills.

microbiology↗

bioRxiv2021

Nanoscale organization of the endogenous ASC speck

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to the formation of a supramolecular assembly of the inflammasome adaptor protein ASC, commonly referred to as the "ASC speck". Different models of the overall structure of the ASC speck, as well as the entire NLRP3 inflammasome, have been reported in the literature. While many experiments involve overexpression or in vitro reconstitution of recombinant ASC, the cytoplasmic endogenous ASC speck remains difficult to study due to its relatively small size and structural variability. Here, we use a combination of fluorescence imaging techniques including dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT) to visualize the endogenous ASC speck following NLRP3 inflammasome activation. We observe that the complex varies in diameter between [~]800 and 1000 nm and is composed of a dense core from which filaments extend into the periphery. We used a combination of anti-ASC antibodies as well as much smaller nanobodies for labeling, and find that the larger antibodies reliably label the lower-density periphery whereas the nanobody, which has a lower binding affinity, is more efficient in labeling the dense core. Imaging whole cells using dSTORM, furthermore, allowed us to sort the imaged structures into a pseudo-time sequence suggesting that the endogenous ASC speck becomes denser but not much larger during its formation. The reported results add an important piece of information towards a comprehensive understanding of the supramolecular structure of the endogenous inflammasome complex. SignificanceThe assembly of ASC into a supramolecular complex referred to as the ASC speck is a critical step in the NLRP3 inflammasome response. Despite significance research, the organization and formation of the endogenous ASC speck remain elusive. We used a combination of differently sized ASC labels with super-resolution microscopy to address the organization of the ASC speck at the nanoscale. Our results allow inference of the supramolecular structure of the unperturbed, endogenous speck and characterize how it changes during ASC recruitment. We find that the complex varies in size and is composed of a dense core from which filaments extend into the periphery. Notably, our results further indicate that during inflammasome formation, ASC specks become denser but only slightly larger.

immunology↗

bioRxiv2020

Virtual-SMLM, a virtual environment for real-time interactive SMLM acquisition

Although single molecule localisation microscopy enables for the visualisation of cells nanoscale organisation, its dissemination remains limited mainly due to the complexity of the associated imaging acquisition, impacting on outputs reliability and reproducibility. We propose here the first all-in-one fully virtual environment for SMLM acquisition: Virtual-SMLM, including on-the-fly interactivity and real time display. It relies on a novel realistic approach to simulate fluorophores photo-physics based on independent pseudo-continuous emission traces. It also facilitates for user-specific experimental and optical environment design. As such, it constitutes a unique tool for the training of both users and machine learning approaches to automated SMLM, as well as for experimental validation, whilst providing realistic data sets for the development of image reconstruction algorithms and data analysis software.

biophysics↗

bioRxiv2020

Nanoscale pattern extraction from relative positions of sparse 3D localisations

We present a method for extracting high-resolution ordered features from localisation microscopy data by analysis of relative molecular positions in 2D or 3D. This approach allows pattern recognition at sub-1% protein detection efficiencies, in large and heterogeneous samples, and in 2D and 3D datasets. We used this method to infer ultrastructure of the nuclear pore, the cardiomyocyte Z-disk, DNA origami structures and the centriole.

cell biology↗

bioRxiv2018

Influenza A viruses use multivalent sialic acid clusters for cell binding and receptor activation

Influenza A virus (IAV) binds its host cell using the major viral surface protein hemagglutinin (HA). HA recognizes sialic acid, a plasma membrane glycan that functions as the specific primary attachment factor (AF). Since sialic acid alone cannot fulfill a signaling function, the virus needs to activate downstream factors to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase family, was shown to be activated by and transmit IAV entry signals. However, how IAV engages and activates EGFR remains largely unclear.\n\nWe used multicolor super-resolution microscopy to study the lateral organization of both IAV attachment factors and its functional receptor at the scale of the IAV particle. Intriguingly, quantitative cluster analysis revealed that AF and EGFR are organized in partially overlapping submicrometer clusters in the apical plasma membrane of A549 cells. Within AF domains, which are distinct from microvilli, the local AF concentration, a parameter that directly influences virus-cell binding, reaches on average 10-fold the background concentration and tends to increase towards the cluster center, thereby representing a multivalent virus-binding platform. Using our experimentally measured cluster characteristics, we simulated virus diffusion on a membrane, revealing that the distinct mobility pattern of IAVs is dominated by the local AF concentration, consistent with live cell single-virus tracking data. In contrast to AF, EGFR resides in clusters of rather low molecular density. Virus binding activates EGFR, but interestingly, this process occurs without a major lateral EGFR redistribution, instead relying on activation of pre-formed clusters, which we show are long-lived.\n\nTaken together, our results provide a quantitative understanding of the initial steps of influenza virus infection. Co-clustering of AF and EGFR permit a cooperative effect of binding and signaling at specific platforms, and thus we relate their spatial organization to their functional role during virus-cell binding and receptor activation.\n\nAuthor SummaryThe plasma membrane is the major interface between a cell and its environment. It is a complex and dynamic organelle that needs to protect as a barrier but also process subtle signals into and out of the cell. For IAV, an enveloped virus, it represents a major obstacle that it needs to overcome during infection as well as the site for the assembly of progeny virus particles. However, the organisation of the plasma membrane in particular the sites of virus interaction at the scale of an infecting particle (length scales < 100 nm) remains largely unknown.\n\nSialic acids serve as IAV attachment factors but are not able to transmit signals across the plasma membrane. Receptor tyrosine kinases were identified to be activated upon virus binding and serve as functional receptor. How IAV engages and activates its functional receptors still remains speculative. Here we use super resolution microscopy to study the lateral organization as well as the functional relationship of plasma membrane-bound molecules involved in IAV infection. We find that molecules are organized in submicrometer nanodomains and, in combination with virus diffusion simulations, present a mechanistic view for how IAV first engages with AFs in the plasma membrane to then engage and trigger entry-associated membrane receptors.

microbiology↗

bioRxiv2018

Multicolor single particle reconstruction of protein complexes

Single-particle reconstruction (SPR) from electron microscopy images is widely used in structural biology, but lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and co-aligns multiple proteins from 2D super-resolution fluorescence images. We demonstrate our method by generating multi-color 3D reconstructions of several proteins within the human centriole and procentriole, revealing their relative locations, dimensions and orientations.

cell biology↗

bioRxiv2018

Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-{kappa}B (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an \"enhanceosome,\" nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.\n\nAuthor SummaryTo control transcription of a certain gene or a group of genes, both eukaryotes and prokaryotes express specialized proteins, transcription factors (TFs). During gene activation, TFs bind gene promotor sequences to recruit the transcriptional machinery including DNA polymerase II. TFs are often multi-subunit proteins containing a DNA-binding domain (DBD) as well as a protein-protein interaction interface. It was suggested that the duration of a TF-DNA binding event 1) depends on these two subunits and 2) dictates the outcome, i.e. the amount of mRNA produced from an activated gene. We set out to address these hypotheses using the transcriptional activator NF-{kappa}B (p65) as well as a number of mutants affecting different functional subunits. Using a combination of live-cell microscopy and RNA sequencing, we show that p65 DNA-binding time indeed correlates with the transcriptional output, but that this relationship depends on, and hence can be uncoupled by altering, the protein-protein interaction capacity. Our results suggest that, while p65 DNA binding times are dominated by the DBD, a transcriptional output can only be achieved with a functional protein-protein interaction subunit.

genetics↗

bioRxiv2017

Relevance of host cell surface glycan structure for cell specificity of influenza A virus

Influenza A viruses (IAV) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycan receptors on host cells. HAs receptor specificity towards sialic acid (SA) is well studied and clearly critical for virus infection, but the contribution of the highly complex cellular plasma membrane to the cellular specificity remains elusive. In addition, some studies indicated that other host cell factors such as the epidermal growth factor receptor might contribute to the initial virus-cell contact and further downstream signaling1.\n\nHere we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS) to compare influenza virus receptor specificity with actual host cell binding. Unexpectedly, our study reveals that HAs receptor binding preference does not necessarily reflect virus-cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAV thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.

microbiology↗