Biology subjects

Carter, S. D.

Publications and source records attributed to Carter, S. D..

10 recordsLinked to original sources

bioRxiv2026

In situ molecular architecture of PML bodies reveals open-state columnar trinucleosome assemblies within a porous, chromatin-permissive interior

Genome function in the nucleus is organised through membrane-less compartments enriched with specific proteins and nucleic acids. Promyelocytic leukemia (PML) bodies regulate telomere maintenance and DNA damage responses, but their internal molecular organisation remains poorly understood. We visualised the molecular composition of PML bodies directly within the nucleus using an advanced cryogenic-correlative light and electron microscopy (cryo-CLEM) pipeline. Cryo-electron tomography revealed eYFP-PML-I bodies as compartments with a molecular makeup distinct from the surrounding nucleoplasm. Cryogenic super-resolution correlative light and electron microscopy showed these comprise a diffuse PML-protein shell enclosing an inner core. A visual proteomics comparison of the core and surrounding regions through template matching and sub-tomogram averaging mapped nucleosomes, TRiC chaperonin complexes in both closed and open conformations, and single-capped PA28 proteasomes. Membrane structures of unknown function were additionally detected within some eYFP-PML-I bodies. Remarkably, the nucleosomes included density consistent with columnar trinucleosome assemblies (1), revealing that PML body interiors harbour discrete chromatin domains. Moreover, vault complexes were detected in poised positions in the proximal nucleoplasm. Together, these first in situ higher-resolution structural insights of PML bodies identify their critical functional components and an interior porous architecture that suggests a mechanism for content selection based on physical partitioning akin to size-exclusion chromatography. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/729032v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19ad697org.highwire.dtl.DTLVardef@7e8590org.highwire.dtl.DTLVardef@72f0caorg.highwire.dtl.DTLVardef@1e8a1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

On-lamella super-resolution cryo-CLEM for cryo-ET enabled by vacuum-free ultra-stable cryogenic fluorescence microscopy

Cryogenic correlative light and electron microscopy (cryo-CLEM) combines specific fluorescence labelling of proteins inside cells with structural information at the angstrom-level. The introduction of super-resolution fluorescence methods in the field of cryogenic fluorescence microscopy is a necessary step to bridge the large resolution gap between the different imaging modalities. However, there are many challenges hindering the full potential of cryogenic super-resolution correlative light and electron microscopy and seamless integration with structural cell biology. One of the main limiting factors is a lack of dedicated cryogenic fluorescence microscopy systems with sufficient mechanical stability to enable the collection of high-quality super-resolution data and full compatibility with vitrified specimens for cryo-electron tomography. Here, we address this by developing a vacuum-free ultra-stable cryogenic optical microscope (VULCROM). VULCROM is a dedicated super-resolution cryo-CLEM (cryo-SR-CLEM) setup that combines the stability of a vacuum-insulated cryostat with the flexibility and modularity of an open microscopy system. We demonstrate that VULCROM enables detailed investigations of single-molecule cryo-photo-physics across timescales spanning milliseconds to hours. We furthermore demonstrate its suitability for routine cryo-SR-CLEM with a resolution in the 10 nm range in distinct vitrified biological specimen types. We resolve the nanoscale architecture of YFP-labelled PML bodies within the nucleus of mammalian cells and the distribution of ATG9-eGFP in its cellular structural context in a cryo-lift-out lamella of N. benthamiana plant tissue. Owing to its vacuum-free design, VULCROM can be readily adapted for diverse correlative workflows and other cryo-light microscopy applications.

biophysics↗

bioRxiv2025

Live-cell 3D-SIM of Rift Valley fever virus NSs filaments reveals a polygonal web architecture

A defining feature of Rift Valley fever virus (RVFV) is the incorporation of the NSs protein into large filamentous assemblies inside infected nuclei 1, as judged from fixed specimens. To gain insight into the three-dimensional (3D) structure of NSs filaments within nuclei of live-cells we used genetic-code expansion (GCE) to introduce an unnatural amino acid (uAA) fluorophore into the protein, and coupled this with live-cell structured illumination microscopy (SIM). Our super-resolved images revealed a micron-scale polygonal web of NSs fibres with discrete domain characteristics. Parallel experiments on fixed RVFV-infected cells confirmed that virally-encoded native NSs filaments also display this morphology. Overall, our 3D-SIM analysis sheds new light on the complex large-scale architecture of NSs filaments and provides context for programmable filamentous E3 ligases that promote virus replication. SignificanceThis work demonstrates how advanced labeling and imaging techniques can reveal unexpected organizational principles in viral protein assemblies. We bio-orthogonally labeled the Rift Valley fever virus NSs protein with bright tetrazine dyes using genetic-code expansion (GCE), that enabled imaging of NSs filaments in live-cells using 3D-SIM. GCE offers key advantages for studying small, genetically fragile viral proteins like NSs. It eliminates the need for bulky fluorescent proteins that can disrupt protein-protein interfaces critical for complex assembly and avoids fixation artifacts associated with traditional immunofluorescence microscopy. This integrative GCE and 3D-SIM approach revealed the complete native architecture of NSs filaments in 3D, dramatically enhancing our ability to visualize intricate polygonal web structures beyond simple linear filaments. The discrete structural domains identified here raise intriguing questions about potential mechanisms for spatially controlling E3 ligase activity in a programmable manner.

biochemistry↗

bioRxiv2024

Ribosome-Associated Vesicles promote activity-dependent local translation

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its deployment to local translation sites remain unclear. Here, we employed a multi-scale imaging approach combining cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify endoplasmic reticulum-derived Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes and promoting activity-dependent local translation. We demonstrate that neuronal stimulation triggers compartment-specific RAV responses: dendrites accumulate stationary RAVs at sites of enhanced translation, while axons accelerate RAV transport. Real-time imaging of translation at single mRNA resolution reveals that RAVs boost local translation output compared to RAV-independent mechanisms. These findings establish RAVs as specialized platforms that integrate activity-dependent signals with local protein synthesis, providing a mechanistic framework for understanding how neurons achieve precise spatiotemporal control of protein synthesis.

neuroscience↗

bioRxiv2023

Structure and Function of the Dot/Icm T4SS

The Legionella pneumophila Dot/Icm type IV secretion system (T4SS) delivers effector proteins into host cells during infection. Despite its significance as a potential drug target, our current understanding of its atomic structure is limited to isolated subcomplexes. In this study, we used subtomogram averaging and integrative modeling to construct a nearly-complete model of the Dot/Icm T4SS accounting for seventeen protein components. We locate and provide insights into the structure and function of six new components including DotI, DotJ, DotU, IcmF, IcmT, and IcmX. We find that the cytosolic N-terminal domain of IcmF, a key protein forming a central hollow cylinder, interacts with DotU, providing insight into previously uncharacterized density. Furthermore, our model, in combination with analyses of compositional heterogeneity, explains how the cytoplasmic ATPase DotO is connected to the periplasmic complex via interactions with membrane-bound DotI/DotJ proteins. Coupled with in situ infection data, our model offers new insights into the T4SS-mediated secretion mechanism.

biochemistry↗

bioRxiv2021

Montage electron tomography of vitrified specimens

Cryo-electron tomography provides detailed views of macromolecules in situ. However, imaging a large field of view to provide more cellular context requires reducing magnification during data collection, which in turn restricts the resolution. To circumvent this trade-off between field of view and resolution, we have developed a montage data collection scheme that uniformly distributes the dose throughout the specimen. In this approach, sets of slightly overlapping circular tiles are collected at high magnification and stitched to form a composite projection image at each tilt angle. These montage tilt-series are then reconstructed into massive tomograms with a small pixel size but a large field of view. For proof-of-principle, we applied this method to the thin edge of HeLa cells. Thon rings to better than 15 [A] were observed in the montaged tilt-series, and diverse cellular features were evident in the resulting tomograms. These results indicate that the additional dose required by this technique is not prohibitive to performing structural analysis to intermediate resolution across a large field of view. We anticipate that montage tomography will prove particularly useful for lamellae, increase the likelihood of imaging rare cellular events, and facilitate visual proteomics.

biophysics↗

bioRxiv2021

Generation of chimeric respiratory viruses with altered tropism by coinfection of influenza A virus and respiratory syncytial virus

Interactions between respiratory viruses impact viral transmission dynamics and clinical outcomes. To identify and characterize virus-virus interactions at the cellular level, we coinfected human lung cells with influenza A virus (IAV) and respiratory syncytial virus (RSV). Super-resolution microscopy, live-cell imaging, scanning electron microscopy, and cryo-electron tomography revealed extracellular and membrane-associated filamentous structures consistent with hybrid viral particles (HVPs). We show that HVPs harbor surface glycoproteins and ribonucleoproteins of IAV and RSV, and use the RSV fusion glycoprotein to evade anti-IAV neutralising antibodies and to infect and spread among cells lacking IAV receptors. Finally, we show evidence of IAV and RSV coinfection within cells of the bronchial epithelium, with viral proteins from both viruses co-localising at the apical surface. Our observations have profound implications for infection biology as they define a previously unknown interaction between respiratory viruses that might affect virus pathogenesis by expanding virus tropism and facilitating immune evasion.

microbiology↗

bioRxiv2021

The stress-sensing domain of activated IRE1α forms helical filaments in narrow ER membrane tubes

The signaling network of the unfolded protein response (UPR) adjusts the protein folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1, spans the ER membrane and activates through oligomerization. IRE1 oligomers accumulate in dynamic foci. We determined the in-situ structure of IRE1 foci by cryogenic correlated light and electron microscopy (cryo-CLEM), combined with electron cryo-tomography (cryo-ET) and complementary immuno-electron microscopy. IRE1 oligomers localize to a network of narrow anastomosing ER tubes (diameter ~28 nm) with complex branching. The lumen of the tubes contains protein filaments, likely composed of linear arrays of IRE1 lumenal domain dimers, arranged in two intertwined, left-handed helices. Our findings define a previously unrecognized ER subdomain and suggest positive feedback in IRE1 signaling.

cell biology↗

bioRxiv2019

Correlated cryogenic fluorescence microscopy and electron cryotomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo

Members of the TRIM protein family have been shown to gather into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy (cryo-CLEM) to intact mammalian cells expressing YFP-rhTRIM5 and found hexagonal nets were present whose arm-lengths were similar to those of the hexagonal nets formed by purified TRIM5 in-vitro. We also observed YFP-rhTRIM5 within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within a double-membraned vesicle (autophagosomes), and sequestosomes within multi-vesicular autophagic vacuoles (autolysosomes or amphisomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data (i) support recent reports that TRIM5 can form both well-organized signaling complexes and non-signaling aggregates, (ii) offer the first images of the macroautophagy pathway in a near-native state, and (iii) reveal that vaults arrive early in macroautophagy.

cell biology↗

bioRxiv2017

Distinguishing Signal From Autofluorescence In Cryogenic Correlated Light And Electron Microscopy Of Mammalian Cells

Cryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processes in situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.

cell biology↗