Biology subjects

Plückthun, A.

Publications and source records attributed to Plückthun, A..

3 recordsLinked to original sources

bioRxiv2025

Pre-assembly of biomolecular condensate seeds drives RSV replication

During infection many RNA viruses, including respiratory syncytial virus (RSV), form specialized biomolecular condensates, inclusion bodies (IBs), where viral transcription and replication occur1-4. Paradoxically, high protein concentrations are typically required for condensate nucleation5, yet attaining sufficient protein levels in infection is thought to require IBs for viral transcription and replication. To uncover how viruses solve this paradox to establish IBs, we visualized early infection of RSV in real-time with single genomic viral ribonucleoprotein (vRNP) resolution. Our results reveal that IBs are nucleated from infecting vRNPs rather than de novo in the cytoplasm. IB nucleation further requires in-virion pre-assembly of viral protein-protein interaction networks on vRNPs to form pre-replication centers (PRCs). PRCs are potent condensate nucleation seeds due to their resistance to disassembly and efficient recruitment of newly-synthesized viral proteins. The high protein affinity of PRCs also results in increased polymerase complex association, allowing efficient viral transcription even in the absence of IBs. Together, these activities create a feed-forward loop that drives rapid IB formation. Intriguingly, PRC assembly depends on in-virion viral protein levels and is highly heterogeneous among virions, explaining cell-to-cell heterogeneity in infection progression, and identifying heterogeneous virions as the origin of infection heterogeneity. Together, our results show that in-virion pre-assembly of PRCs kick-starts viral condensate nucleation upon host-cell entry, and explains cell-to-cell heterogeneity in RSV infection.

microbiology↗

bioRxiv2022

A Vaccinia-based system for directed evolution of GPCRs in mammalian cells

Low stability and poor expression levels are inherent in many G protein-coupled receptors, hindering structural and biophysical analyses. Directed evolution in bacterial or yeast display systems has been successfully used to overcome both limitations in some cases. Yet, some receptors cannot be tackled in microbial systems, due to their complex molecular composition or due to unfavorable ligand properties. Here, we report an approach to evolve G protein-coupled receptors in mammalian cells. To achieve clonality of individual mutants, and a rather uniform gene copy number, both prerequisites for strict genotype-phenotype linkage in mammalian cells, we developed a highly efficient viral transduction system based on Vaccinia virus. Using rational design of synthetic DNA libraries, we first evolved neurotensin receptor 1 for high stability and expression, reaching levels similar or higher to what has been achieved in bacterial systems. Second, using parathyroid hormone 1 receptor we demonstrate that also receptors with complex molecular architectures and large ligands can be readily evolved in mammalian cells. Furthermore, in our system, the physiological signaling environment in mammalian cells can be employed to evolve functional receptor properties. We obtained fully functional receptor variants exhibiting increased allosteric coupling between the ligand binding site and the G protein interface, resulting in higher signaling efficacy. Thus, our approach provides new means to readily improve the biophysical properties of receptors in a mammalian cellular environment. Moreover, it opens the possibility to modulate receptor signaling and to gain further insights into the intricate molecular interplay required for GPCR activation.

biochemistry↗

bioRxiv2022

A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

Neuroscience currently requires the use of antibodies to study synaptic proteins, where antibody binding is used as a correlate to define the presence, plasticity, and regulation of synapses. Gephyrin is an inhibitory synaptic scaffolding protein used to mark GABAergic and glycinergic postsynaptic sites. Despite the importance of gephyrin in modulating inhibitory transmission, its study is currently limited by the tractability of available reagents. Designed Ankyrin Repeat Proteins (DARPins) are a class of synthetic protein binder derived from diverse libraries by in vitro selection, and tested by high-throughput screening to produce specific binders. In order to generate a functionally diverse toolset for studying inhibitory synapses, we screened a DARPin library against gephyrin mutants representing both phosphorylated and dephosphorylated states. We validated the robust use of anti-gephyrin DARPin clones for morphological identification of gephyrin clusters in rodent neuron culture and brain tissue, discovering previously overlooked clusters. This DARPin-based toolset includes clones with heterogenous gephyrin binding modes that allowed for identification of the most extensive gephyrin interactome to date, and defined novel classes of putative interactors, creating a framework for understanding gephyrins non-synaptic functions. This study demonstrates anti-gephyrin DARPins as a versatile platform for studying inhibitory synapses in an unprecedented manner.

neuroscience↗