Biology subjects

O'Sullivan, T. J.

Publications and source records attributed to O'Sullivan, T. J..

3 recordsLinked to original sources

bioRxiv2025

The architecture of membrane structures involved in hepatitis C virus genome replication revealed in close-to-native conditions by cryo-electron tomography

Hepatitis C virus (HCV) infection induces extensive rearrangements of host cytoplasmic membranes, leading to the formation of multiple membranous structures that facilitate RNA replication. Current knowledge of these membranous structures has largely relied on correlative light and electron microscopy (CLEM) techniques using chemical fixation and resin embedding. To overcome these limitations, cryo-preserved cells were prepared using cryo-focused ion beam (cryo-FIB) milling and cryo-ultramicrotomy. For the first time, the contents within the membranous structures have been observed in-situ using cryo-electron tomography (cryo-ET) performed on lamellae (prepared via cryo-FIB) and on ultrathin sections (prepared via cryo-ultramicrotomy) from HCV subgenomic replicon harbouring cells. Observations from 112 cryo-electron tomograms of cryo-FIB-derived samples revealed the presence of densities within the inner vesicles of a subset of single-, double-membrane vesicles (SMVs, and DMVs respectively), as well as within multi-vesicular bodies (MVBs), which might represent the viral replication machinery. Notably, this study represents the first direct visualisation of the arrangement of non-structural proteins within a multi-membrane vesicle (MMV) observed from cryo-electron microscopy of vitreous sections (CEMOVIS). The cryo-ET methodologies established here lay the groundwork for future investigations into the architecture of the HCV replication complex, leveraging advanced computational tools for deeper structural and functional analysis.

microbiology↗

bioRxiv2025

Memory engram synapse 3D molecular architecture visualized by cryoCLEM-guided cryoET

Memory is incorporated into the brain as physicochemical changes to engram cells. These are neuronal populations that form complex neuroanatomical circuits, are modified by experiences to store information, and allow for memory recall. At the molecular level, learning modifies synaptic communication to rewire engram circuits, a mechanism known as synaptic plasticity. However, despite its functional role on memory formation, the 3D molecular architecture of synapses within engram circuits is unknown. Here, we demonstrate the use of engram labelling technology and cryogenic correlated light and electron microscopy (cryoCLEM)-guided cryogenic electron tomography (cryoET) to visualize the in-tissue 3D molecular architecture of engram synapses of a contextual fear memory within the CA1 region of the mouse hippocampus. Engram cells exhibited structural diversity of macromolecular constituents and organelles in both pre- and postsynaptic compartments and within the synaptic cleft, including in clusters of membrane proteins, synaptic vesicle occupancy, and F-actin copy number. This engram to tomogram approach, harnessing in vivo functional neuroscience and structural biology, provides a methodological framework for testing fundamental molecular plasticity mechanisms within engram circuits during memory encoding, storage and recall.

neuroscience↗

bioRxiv2023

In situ cryo-electron tomography of beta-amyloid and tau in post-mortem Alzheimer's disease brain

A defining pathological feature of most neurodegenerative diseases is the assembly of proteins into amyloid that form disease-specific structures. In Alzheimers disease (AD) this is characterised by the deposition of amyloid-{beta} (A{beta}) and tau with AD-specific conformations. The in situ structure of amyloid in the human brain is unknown. Here, using cryogenic fluorescence microscopy (cryoFM)-targeted cryo-sectioning, cryo-focused ion beam scanning electron microscopy (cryoFIB-SEM) liftout and cryo-electron tomography (cryoET), we determined the in-tissue structure of {beta}-amyloid and tau pathology in fresh post-mortem AD donor brain. {beta}-amyloid plaques contained a mixture of fibrils and protofilaments arranged in parallel arrays and lattice-like structures, some of which were branched. Extracellular vesicles, extracellular droplets and open lipid bilayer sheets defined non-amyloid constituents of amyloid plaques. In contrast, tau inclusions were characterised by clusters of unbranched filaments. Subtomogram averaging of filaments within each cluster revealed distinct structures including variably twisted paired helical filaments (PHF) and chronic traumatic encephalopathy (CTE)-like tau filaments that were situated [~]1 m apart within two microscopic regions of pathology. Filaments within a cluster were similar to each other, but different between clusters, showing that fibril heterogeneity is spatially organised and influenced by the subcellular tissue environment. The in situ structural approaches outlined here for targeting specific proteins within human donor tissues have applications to a broad range of neurodegenerative diseases.

pathology↗