Biology subjects

Beck, F.

Publications and source records attributed to Beck, F..

7 recordsLinked to original sources

bioRxiv2025

Nanoscale imaging of native symbiotic animal tissue using amultimodal large volume imaging pipeline for cryo-electrontomography

The field of cryo-EM offers the possibility to gain high-resolution structural information of biomolecules in their native state. Advances in sample thinning of cryo-EM samples allows the study of proteins inside intact cells using tomography, opening the door for visual proteomics. However, thicker samples such as tissues or entire organisms are still largely unsuitable for cryo-electron tomography (cryo-ET). Therefore, significant efforts are directed toward developing and improving preparation methods to enable cryo-ET of such complex samples. We focused on the binary association between the Hawaiian bobtail squid Euprymna scolopes and the luminous bacteria Vibrio fischeri. The Squid-Vibrio system has long been studied to understand host-symbiont interactions. Our goal is to study the bacterial-host interface using cryo-ET, at a resolution previously unattainable by conventional EM methods. Here, we present a multi-modal preparation and correlative imaging workflow--including cryo- fluorescence microscopy, microCT, freeze-substitution electron tomography (FS-ET), and serial blockface SEM--to localize and prepare specific regions of the dissected symbiotic light organs for cryo-ET. This approach enabled us to directly visualize symbiotic V. fischeri within the internal host crypts at macromolecular resolution, revealing spatial organization, physical contact, and putative exchange interfaces between host and microbe. Our findings provide structural insights into a foundational model of host-microbe symbiosis and demonstrate the feasibility of cryo-ET for investigating intact tissues at the nanoscale.

microbiology↗

bioRxiv2025

The targeting of non-fibrillar polyQ via distinct VCP-proteasome coupling

The aggregation of proteins containing expanded poly-glutamine (polyQ) repeat sequences is a cytopathological hallmark of several dominantly inherited neurodegenerative diseases (ND), including Huntingtons disease (HD). Previously, we observed that long polyQ repeats form a fibrillar core, surrounded by amorphous and soluble intermediates, which compared to the fibrils, are more readily taken up by autophagosomes 1. In this study, we investigated how the alternative major degradation pathway in cultured cells, ubiquitin proteasome system (UPS), interacts with the different pools of polyQ. We observed that the AAA+ ATPase, valosin-containing protein VCP/p97, in collaboration with proteasomes, plays a crucial role in degrading non-fibrillar polyQ. As both VCP and proteasomes were recruited to the polyQ intermediates peripheral to the fibrils, we imaged these regions by in situ cryo-ET for subtomogram averaging. VCP predominantly adopts an ATP-bound state, often in an active processing conformation with resolved NPLOC4-like cofactor density. The region was also enriched with the 26S proteasome (26S), 20S proteasome core (20S), and 19S regulatory particles (19S). Distance analysis of the macromolecules revealed a striking proximity between VCP and the 20S in situ. This is likely mediated by VCPs C-terminal hydrophobic-tyrosine-X (HbYX) motif, which binds the 20S via hydrophobic interactions, as confirmed by cryo-EM single-particle analysis (SPA) and in vitro assays. Our study highlights a distinct VCP-20S coupling mechanism, where VCP functionally overlaps with the 19S complex in substrate unfolding, facilitating the degradation of polyQ intermediates by the 20S catalytic core.

biochemistry↗

bioRxiv2024

In-cell structure and snapshots of copia retrotransposons in intact tissue by cryo-electron tomography

Long terminal repeat (LTR) retrotransposons belong to the transposable elements (TE), autonomously replicating genetic elements that integrate into the hosts genome. LTR retrotransposons represent a major component of genomes across the tree of life; some derived sequences have even been domesticated by the host to perform cellular functions in essential processes such as development. Among animals, Drosophila melanogaster serves as an important model organism for TE research, harboring several LTR retrotransposons, including the Ty1-copia family, which is evolutionarily related to retroviruses and forms virus-like particles (VLPs). The architectural organization of copia VLPs in situ has remained unknown. In this study, we use cryo-FIB milling and lift-out approaches to visualize copia VLPs in isolated ovarian cells and intact egg chambers and resolve the in situ copia capsid structure to 7.7 [A] resolution by cryo-ET. While cytosolic copia VLPs vary in size, nuclear VLPs are homogenous and form densely packed clusters, supporting a model in which nuclear import acts as a size selector. By analyzing flies deficient in the TE-suppressing PIWI-piRNA pathway, we observe a change in copia localization from cytosolic to nuclear during spermatogenesis in testes. Our findings provide insights into the cellular structural biology of an active LTR retrotransposon and shed light on the replication cycle of copia in the context of host gametogenesis.

biophysics↗

bioRxiv2023

Serial Lift-Out - Sampling the Molecular Anatomy of Whole Organisms

Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically < 300 nm. Here, we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to C. elegans L1 larvae yielding a cryo-ET dataset sampling the worms anterior-posterior axis and resolve its ribosome structure to 7 [A], illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.

biophysics↗

bioRxiv2022

Cryo-electron tomography reveals enrichment and identifies microtubule lumenal particles in neuronal differentiation

The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most of which are known to bind to the MT outer surface. However, electron microscopy (EM) has long ago revealed the presence of particles inside the lumens of neuronal MTs, of yet unknown identity and function. Here, we use cryogenic electron tomography (cryo-ET) to analyze the three-dimensional (3D) structures and organizations of MT lumenal particles in primary hippocampal neurons, human induced pluripotent stem cell-derived neurons and pluripotent P19 cells. We obtain in-cell 3D maps of several lumenal particles from the respective cells and detect structural features that are common to all cell-types, underscoring their potential overarching functions. Mass spectrometry-based proteomics combined with structural modeling suggests a subset of lumenal particles could be tubulin-binding cofactors (TBCs) bound to tubulin monomers. A different subset of smaller particles, which remains unidentified, exhibits densities that bridge across the MT protofilaments. We show that increased lumenal particle concentration within MTs is concomitant with neuronal differentiation and correlates with higher MT curvatures. Enrichment of lumenal particles around MT lattice defects and at freshly polymerized MT open-ends suggest a MT protective role. Together with the identified structural resemblance of a subset of particles to TBCs, these results hint at a role in local tubulin proteostasis for the maintenance of long-lived neuronal MTs.

cell biology↗

bioRxiv2022

Optimizing Cryo-FIB Lamellas for sub-5 Angstrom in situ Structural Biology

We here present a method based on metallic platinum sputtering that can substantially enhance the quality of subtomogram averages from lamellas and thereby reduce the number of particles needed for high-resolution subtomogram averaging. We provide evidence for the physical background of this improvement and demonstrate its usefulness by producing sub-5[A] ribosome averages from yeast.

molecular biology↗

bioRxiv2022

In situ structural analysis reveals membrane shape transitions during autophagosome formation

Autophagosomes are unique organelles which form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves a series of membrane transformations with distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (ET) and novel data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. By mapping individual structures onto a timeline based on geometric features, we uncover dynamic changes in membrane shape and curvature. Moreover, we reveal the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles such as the vacuole and the ER. Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores towards closure without a templating cargo.

cell biology↗