Biology subjects

Moravcova, J.

Publications and source records attributed to Moravcova, J..

3 recordsLinked to original sources

bioRxiv2026

Automated cryo-volume EM for high-resolution 3D imaging and in situ structural analysis of cells and tissues

Cryo-volume electron microscopy (CVEM) enables three-dimensional imaging of biological ultrastructure in a near-native state but has been limited by low image contrast and charging artifacts that hinder data interpretation and complicate automation of data acquisition. Here we present an experimental and computational workflow that combines orthogonal cryo-SEM imaging, spot-geometry optimized O+ plasma-FIB milling, dedicated acquisition-control routines, and dedicated image alignment procedure. The workflow enables autonomous acquisition of volumetric datasets from vitrified cells and tissues at [~]15-20 nm isotropic resolution. In addition, sub-volume averaging of 113 nuclear pore complexes extracted from CVEM dataset of Cos-7 cell yielded its reconstruction at 9.4 nm resolution. Together, these results establish CVEM as a robust platform for autonomous high-resolution volumetric imaging and structural analysis of vitrified biological specimens.

Cell Biology↗

bioRxiv2023

Cryo-electron tomography of enterovirus cell entry and endosome escape

Enveloped viruses deliver their genomes into the cell cytoplasm by membrane fusion; in contrast, membrane penetration by non-enveloped viruses is more diverse and less well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. To initiate infection, most enteroviruses enter cells by endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing rhinovirus 2, echovirus 18, echovirus 30, or enterovirus 71 deform, rupture, and release their content into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of enterovirus particles that released their genomes in endosomes, but did not prevent them from reaching the cytoplasm. Inhibiting N-WASP-mediated post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. We show that the rupture of endosomes also occurs in uninfected cells. In summary, our results indicate that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus genomes and particles into the cytoplasm. Since the studied enteroviruses employ different receptors for cell entry but are all delivered into the cytoplasm by cell-mediated endosome disruption, it is possible that many other enteroviruses utilize endosome rupture to infect cells.

microbiology↗

bioRxiv2023

Ultrastructural details of resistance factors of the bacterial spore revealed by in situ cryo-electron tomography

The bacterial spore owes its incredible resistance capacities to various molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography (cryo-ET) on bacteria lamellae generated by cryo-focused ion beam micromachining (cryo-FIBM) provides insights into the ultrastructural organization of Bacillus subtilis sporangia, including that of the DNA and nascent coat layers. Analysis of the reconstructed tomograms reveal that rather early during sporulation, the chromosome in the developing spore (the forespore) adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a complex stack of amorphous or structured layers with distinct electron density, dimensions and organization. We investigated the nature of the nascent coat layers in various mutant strains using cryo-FIBM/ET and transmission electron microscopy on resin sections of freeze-substituted bacteria. Combining these two cellular electron microscopy approaches, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of the morphogenetic proteins SpoIVA, SpoVID, SafA and/or CotE. Significance statementBacterial spores are dormant cells that can resist to multiple stresses, including antibiotics, detergents, irradiation and high temperatures. Such resilience is an asset when spores are used for the benefit of humans, as in the case of probiotics, or a major problem for public health, food safety or biowarfare when it comes to spores of pathogenic bacteria. In this study, we combined state-of-the-art cryo-electron tomography and conventional cellular electron microscopy to provide insights into intermediate stages of spore development. Our data reveal the intracellular reorganization of the chromosome into a toroidal fibrillar structure and the complex assembly of the multi-protein, multilayered extracellular coat, shedding light on the mechanisms by which the spore acquires its incredible resistance capacities.

microbiology↗