Biology subjects

Förster, F.

Publications and source records attributed to Förster, F..

7 recordsLinked to original sources

bioRxiv2026

In-cell structural insights into fungal ER stress responses

Approximately a third of a cells proteome is folded in the endoplasmic reticulum (ER). The accumulation of misfolded proteins in the ER results in ER stress. The unfolded protein response (UPR) has evolved to attenuate ER stress by increasing the cells folding capacity and reducing the folding load in the ER. In metazoans, the latter is notably achieved through mRNA translation inhibition. However, S. cerevisiae lacks the corresponding pathways, and its reliance on translational downregulation in response to ER stress remains largely unexplored. In this study we combined cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to investigate the effect of ER stress on the translational machinery of S. cerevisiae. At the ultrastructural level, we describe changes in organelle morphology, confirming ER volume expansion and the activation of autophagy. Further structural analysis reveals that ER stress triggers an increase in the relative abundance of inactive 80S ribosomes (up to 25% of the total), both in the cytosol and at the ER. Finally, we visualise the binding of the elongation factors eEF2, eIF5A and eEF3 to inactive ribosomes. Our work provides evidence that ER stress in S. cerevisiae leads to modest translation inhibition, which might contribute to alleviating ER stress.

molecular biology↗

bioRxiv2024

pytom-match-pick: a tophat-transform constraint for automated classification in template matching

Template matching (TM) in cryo-electron tomography (cryo-ET) enables in situ detection and localization of known macromolecules. However, TM faces challenges such as interfering features with a high signal-to-noise ratio and the need for manual curation of results. To address these challenges, we introduce pytom-match-pick, a GPU-accelerated, open-source command line interface for enhanced TM in cryo-ET. Using pytom-match-pick, we first quantify the effects of point spread function (PSF) weighting and show that a tilt-weighted PSF outperforms a binary wedge with a single defocus estimate. We also assess previously introduced background normalization methods for classification performance. This indicates that phase randomization is more effective than spectrum whitening in reducing false positives. Furthermore, a novel application of the tophat transform on score maps, combined with a dual-constraint thresholding strategy, reduces false positives and improves precision. We benchmarked pytom-match-pick on public datasets, demonstrating improved classification and localization of macromolecules like ribosomal subunits and proteasomes that led to fewer artifacts in subtomogram averages. This tool promises to advance visual proteomics by improving the efficiency and accuracy of macromolecule detection in cellular contexts.

molecular biology↗

bioRxiv2023

Visualization of translation reorganization upon persistent collision stress in mammalian cells

Aberrantly slow mRNA translation leads to ribosome stalling and subsequent collision with the trailing neighbor. Ribosome collisions have recently been shown to act as stress sensors in the cell, with the ability to trigger stress responses balancing survival and apoptotic cell-fate decisions depending on the stress level. However, we lack a molecular understanding of the reorganization of translation processes over time in mammalian cells exposed to an unresolved collision stress. Here we visualize the effect of a persistent collision stress on translation using in situ cryo electron tomography. We observe that low dose anisomycin collision stress leads to the stabilization of Z-site bound tRNA on elongating 80S ribosomes, as well as to the accumulation of an off-pathway 80S complex possibly resulting from collision splitting events. We visualize collided disomes in situ, occurring on compressed polysomes and revealing a stabilized geometry involving the Z-tRNA and L1 stalk on the stalled ribosome, and eEF2 bound to its collided rotated-2 neighbor. In addition, non-functional post-splitting 60S complexes accumulate in the stressed cells, indicating a limiting Ribosome associated Quality Control clearing rate. Finally, we observe the apparition of tRNA-bound aberrant 40S complexes shifting with the stress timepoint, suggesting a succession of different initiation inhibition mechanisms over time. Altogether, our work visualizes the changes of translation complexes under persistent collision stress in mammalian cells, indicating how perturbations in initiation, elongation and quality control processes contribute to an overall reduced protein synthesis. SummaryUsing in situ cryo electron tomography we visualized the reorganization of mammalian translation processes during a persistent collision stress.

cell biology↗

bioRxiv2022

Thriving in the heat - Lysine acetylation stabilizes the quaternary structure of a Mega-Dalton hyperthermoactive PEP-synthase

Over time structural adaptations enabled proteins and enzymes to have sufficient stability and flexibility to perform the basic functions of life under various environmental conditions. The catalytic cores of key metabolic enzymes of hyperthermophilic archaea work at a temperature range of 80-120 {degrees}C, similar to the conditions wher the earliest life forms may have thrived. Here we characterize a key enzyme of the central carbon metabolism of Pyrococcus furious, through an integrative approach combining structural mass spectrometry, cryo-electron microscopy, mass photometry and molecular modelling with molecular dynamics simulations. From our investigation, we unveil the structural organization of phosphoenolpyruvate synthase (PPSA). Its 24-meric assembly - weighing over 2 MDa - harbors flexible distal domains, whose proper functioning and coordination depends on widespread chemical acetylation of lysine residues. This non-enzymatic post-translational modification, along with other types of lysine modifications, also occurs on most other major protein complexes of P. furiosus. These modifications likely originated in the chemically favorable primordial conditions and gradually became highly specialized and enzyme-driven in more distantly related mesophiles and Eukaryotes.

biochemistry↗

bioRxiv2022

Increased microtubule lattice spacing correlates with selective binding of kinesin-1 in cells

Within the cell cargo is transported via motor proteins walking along microtubules. The affinity of motor proteins for microtubules is controlled by various layers of regulation like tubulin isoforms, post- translational modifications and microtubule associated proteins. Recently, the conformation of the microtubule lattice has also emerged as a potential regulatory factor, but to what extent it acts as an additional layer of regulation has remained unclear. In this study, we used cryo-correlative light and electron microscopy to study microtubule lattices inside cells. We find that, while most microtubules have a compacted lattice ([~]41 [A]), a significant proportion of the microtubule cores have expanded lattice spacings and that these lattice spacings could be modulated by the microtubule stabilizing drug Taxol. Furthermore, kinesin-1 predominantly binds microtubules with a more expanded lattice spacing ([~]41.6 [A]). The different lattice spacings present in the cell can thus act as an additional factor that modulates the binding of motor proteins to specific microtubule subsets.

molecular biology↗

bioRxiv2021

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes the hexameric complex

The enterovirus genus encompasses many clinically important human pathogens such as poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These viruses are the etiological agents of several human diseases, including hand-foot-and-mouth disease, neonatal sepsis, encephalitis, meningitis, paralysis and respiratory infections. There is an unmet need for antivirals to treat these diseases. The non-structural protein 2C is a AAA+ helicase and plays a key role in viral replication. As such, it is an attractive target for antiviral drug development. Several repurposing screens with FDA-approved drugs have identified 2C-targeting compounds such as fluoxetine and dibucaine, but the molecular basis of 2C inhibition has remained enigmatic. Here we present the 1.5 [A] resolution crystal structure of the soluble fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), which reveals a conserved, hydrophobic drug-binding pocket which is distal to the ATP binding site. To decipher the molecular mechanism of inhibition by fluoxetine and other 2C-targeting compounds, we engineered a soluble, hexameric and ATPase competent 2C protein. Using this system, we show that SFX, dibucaine, HBB and guanidine hydrochloride inhibit 2C ATPase activity in a dose-dependent manner. Moreover, using cryo-EM analysis, we demonstrate that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition and allowing us to generate the first reconstruction of the oligomeric complex. Taken together, these results provide important structural and mechanistic insights into 2C inhibition and provide a robust engineering strategy which can be used for structural, functional and drug-screening analysis of 2C proteins from current or future enteroviruses.

microbiology↗

bioRxiv2020

A human monoclonal antibody targeting a conserved pocket in the SARS-CoV-2 receptor-binding domain core

SARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.

immunology↗