Biology subjects

Dumoux, M.

Publications and source records attributed to Dumoux, M..

11 recordsLinked to original sources

bioRxiv2026

Adaptive Milling: imaging feedback-driven automated fabrication of consistently thin cryo-lamellae

Cryo-electron tomography of 100-200 nm lamellae prepared by focused ion beam milling enables structural analysis of macromolecules within cells, tissues, and small organisms, but lamella preparation remains time-consuming and limits throughput. Although automated workflows enable unsupervised fabrication, they commonly produce variable results because fixed, pre-calibrated parameters are applied without accounting for lamella-to-lamella variability. To overcome these limitations, we developed an Adaptive Milling approach that adapts milling parameters based on imaging feedback. We implemented this approach for the critical polishing stage as Adaptive Polishing, which uses machine-learning-based segmentation of scanning electron microscopy images to identify lamella features predictive of imminent collapse. These are used to periodically assess whether to continue or stop thinning during polishing. We performed a systematic comparison of the quality of lamellae prepared with Adaptive Polishing and conventional automation, and demonstrate that Adaptive Polishing produces thinner lamellae with greater consistency in thickness, and performs well for in situ cryo-electron tomography. Adaptive Polishing was tested on three different microscopes with minimal model retraining required, demonstrating transferability of the approach. We implemented Adaptive Polishing as an open-source plugin for fibsemOS together with machine-learning model training software and pre-trained models.

molecular biology↗

bioRxiv2026

Spatiotemporal biogenesis of thylakoid membranes in the green alga Chlamydomonas reinhardtii

Thylakoid membranes are essential for oxygenic photosynthesis, yet the mechanisms underlying their spatial and temporal biogenesis remain poorly understood. Here, using a light-induced membrane regeneration system in Chlamydomonas reinhardtii, we generate a time-resolved map of thylakoid formation and photosynthetic complex assembly at unprecedented spatial and temporal resolution by integrating super-resolution fluorescence microscopy, cryo-electron tomography, proteomics, and spectroscopy. We show that thylakoid biogenesis is globally distributed and multipolar, with new membrane formation occurring at multiple sites across the chloroplast, including regions adjacent to the inner envelope membranes in both basal and lobe regions. We identify F-ATP synthase storage membranes and map the redistribution of translating ribosomes from T-zone enrichment to a chloroplast-wide distribution at early stages of thylakoid formation, consistent with decentralized synthesis of photosynthetic complexes. We further delineate the hierarchical assembly of photosystem supercomplexes, starting with formation of reaction centers. Together, these findings refine the spatial organization of photosynthetic membrane biogenesis and establish a unified spatiotemporal framework linking thylakoid formation, translational activation, and functional maturation, providing a mechanistic basis for plastid engineering to enhance photosynthetic efficiency.

Cell Biology↗

bioRxiv2025

Early HIV-1 maturation drives Env clustering and fusion competence

Human immunodeficiency virus type 1 (HIV-1) particles are initially released from the cell in an immature state that cannot cause infection. The proteolytic processing of the Gag structural polyprotein into its individual components triggers a dramatic structural reorganisation that produces fully mature and infectious virions. HIV-1 maturation remodels the Gag lattice to enable membrane fusion, but how early proteolytic steps regulate the envelope glycoprotein (Env) organisation is unknown. Although cryo-electron tomography has defined structural intermediates of maturation, it has not resolved how Env conformers relate to specific early maturation states. Here, we show that partial Gag cleavage, prior to capsid formation, allosterically reorganises the membrane-proximal lattice, promoting Env clustering and fusion-competent conformations. Using native virus preparations combined with cryogenic fluorescence-lifetime imaging correlated to cryo-electron tomography, we map Env conformational signatures onto defined Gag-processing intermediates. This reveals an unrecognised early maturation stage in which incomplete Gag processing primes Env for entry, shifting current models by positioning functional Env activation upstream of core maturation. These results establish that early lattice remodelling is a key determinant of HIV-1 fusion competence and expose a previously inaccessible, intervention-sensitive step with implications for therapeutic and vaccine strategies targeting maturation-entry coupling.

microbiology↗

bioRxiv2025

In-cell Structure and Variability of Pyrenoid Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a key enzyme in the global carbon cycle, catalyzing CO2 fixation during photosynthesis. To overcome Rubiscos inherent catalytic inefficiency, many photosynthetic organisms have evolved CO2-concentrating mechanisms. Central to these mechanisms is the pyrenoid, a protein-dense organelle within the chloroplast of eukaryotic algae, which increases the local concentration of CO2 around Rubisco and thereby enhances its catalytic efficiency. Although the structure of Rubisco has been extensively studied by in vitro methods such as X-ray crystallography and single particle cryo-EM, its native structure within the pyrenoid, its dynamics, and its association with binding partners remain elusive. Here, we investigate the structure of native pyrenoid Rubisco inside the green alga Chlamydomonas reinhardtii by applying cryo-electron tomography (cryo-ET) on cryo-focused ion beam (cryo-FIB) milled cells, followed by subtomogram averaging and 3D classification. Reconstruction at sub-nanometer resolution allowed accurate modeling and determination of a closed (activated) Rubisco conformation. Comparison to other reconstructed subsets revealed local variations at the complex active site and at the large subunit dimers interface, as well as association with binding proteins. The different structural subsets distribute stochastically within the pyrenoid. Taken together, these findings offer a comprehensive description of the structure, dynamics, and functional organization of Rubisco within the pyrenoid, providing valuable insights into its critical role in CO2 fixation.

cell biology↗

bioRxiv2024

Reduction of SEM charging artefacts in native cryogenic biological samples.

Scanning electron microscopy (SEM) of frozen-hydrated biological samples allows imaging of subcellular structures at the mesoscale in their native state. Combined with focused ion beam milling (FIB), serial FIB/SEM can be used to build a 3-dimensional picture of cells and tissues. The correlation of specific regions of interest with cryo-electron microscopy (cryoEM) can additionally enable subsequent high-resolution analysis. However, the adoption of serial FIB/SEM imaging-based methods is limited due to artefacts arising from insulating areas of cryogenically preserved samples. Here, we demonstrate the use of interleaved scanning to reduce charging artefacts, allowing the observation of biological features that otherwise would be masked or perturbed. We apply our method to samples where inherent features are not visible. These examples include membrane contact sites within mammalian cells, visualisation of the degradation compartment in the algae E.gracilis and observation of a network of membranes within different types of axons in an adult mouse cortex. We further propose an alternative scanning method that could also be widely applicable to imaging any non-conductive.

cell biology↗

bioRxiv2024

Xenon plasma focused ion beam lamella fabrication on high-pressure frozen specimens for structural cell biology

Cryo focused ion beam lamella preparation is a potent tool for in situ structural biology, enabling the study of macromolecules in their native cellular environments. However, throughput is currently limited, especially for thicker, more biologically complex samples. We describe how xenon plasma focused ion beam milling can be used for routine bulk milling of thicker, high-pressure frozen samples during lamellae preparation with a high success rate and determine a 4.0 [A] structure of the Escherichia coli ribosome on these lamellae using sub volume averaging. We determine the effects of increased ion currents on sample integrity during bulk milling of thicker planar samples, also showing that beyond an initial region of damage, no measurable structural damage propagates beyond this. The use of xenon results in substantial structural damage to particles up to 30 nm in depth from the milled surfaces, with detectable damage observed to 45 nm. Ours results outlines how the use of high currents using xenon plasma focused ion beam milling may be integrated into FIB milling regimes for preparing thin lamellae for high-resolution in situ structural biology.

biochemistry↗

bioRxiv2022

Okapi-EM - a napari plugin for processing and analysing cryogenic serial FIB/SEM images

An emergent volume electron microscopy (vEM) technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at mesoscale resolution. This is achieved by collecting consecutive SEM images after successive rounds of FIB milling that expose a new surface after each milling step. Due to instrumental limitations, some image processing is necessary before 3D visualisation and analysis of the data is possible. SEM images are affected by noise, drift, and charging effects, that can make precise 3D reconstruction of biological features difficult. This paper presents Okapi-EM, an open-source Napari plugin(1) developed to process and analyse cryogenic serial FIB/SEM images. Okapi-EM enables automated image registration of slices, evaluation of image quality metrics specific to FIB-SEM imaging, and mitigation of charging artefacts. Implementation of Okapi-EM within the Napari framework ensures that the tools are both user- and developer-friendly, through provision of a graphical user interface and access to Python programming. Napari also hosts a variety of other image processing plugins so Okapi-EM tools can be integrated into and combined with other workflows. Okapi-EM can be downloaded freely at https://github.com/rosalindfranklininstitute/okapi-em, or installed from Python package index (PyPI). Impact statementCryogenic serial pFIB/SEM is an emerging microscopy technique that is used to visualise 3D structures of biological features at mesoscale resolutions(2). This technique requires common post processing of data such as alignment and charge mitigation to enable robust segmentation and analysis. In addition, approaches are needed to quantify data quality to enable an assessment of features and tune data acquisition parameters to enable optimal image acquisition. This article presents Okapi-EM, a combination of software tools designed to facilitate these important initial steps in assessing and processing images from these experiments. These tools have been assembled as a plugin for a popular 3D biological image visualiser called Napari, making their usage user-friendly and readily accessible.

bioinformatics↗

bioRxiv2022

Ot2Rec: A Semi-Automatic, Extensible, Multi-Software Tomographic Reconstruction Workflow

Electron cryo-tomography (cryo-ET) is an imaging technique for probing 3D structures with at the nanometre scale. This technique has been used extensively in the biomedical field to study the complex structures of proteins and other macromolecules. With the advancement in technology, microscopes are currently capable of producing images amounting to terabytes of data per day, posing great challenges for scientists as the speed of processing of the images cannot keep up with the ever-higher throughput of the microscopes. Therefore, automation is an essential and natural pathway on which image processing - from individual micrographs to full tomograms - is developing. In this paper, we present Ot2Rec, an open-source pipelining tool which aims to enable scientists to build their own processing workflows in a flexible and automatic manner. The basic building blocks of Ot2Rec are plugins which follow a unified API structure, making it simple for scientists to contribute to Ot2Rec by adding features which are not already available. In this paper, we also present three case studies of image processing using Ot2Rec, through which we demonstrate the speedup of using a semi-automatic workflow over a manual one, the possibility of writing and using custom (prototype) plugins, and the flexibility of Ot2Rec which enables the mix-and-match of plugins. We also demonstrate, in the supplementary information, a built-in reporting feature in Ot2Rec which aggregates the metadata from all process being run, and output them in the Jupyter Notebook and/or HTML formats for quick review of image processing quality. Ot2Rec can be found at https://github.com/rosalindfranklininstitute/ot2rec. Impact StatementThe field of cryo electron tomography has grown substantially in recent years, bringing about new advances in hardware and software which enable visualisation of cell and tissue architecture and proteins found in their native context. These same advances have, in some ways, stratified the field into those with access and those without. On the software side, this has emphasised the need for open-source options that do not require high levels of computational literacy to access. Additionally, it has highlighted the need for ways to both mix-and-match software for easy prototyping and comparisons between parameters and methods. Ot2Rec addresses these needs through a simple, unified plugin structure allowing the addition of existing software or the development of new and does so in a way which democratises access.

bioinformatics↗

bioRxiv2022

Cryo-plasma FIB/SEM volume imaging of biological specimens

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 m). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free tool is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasmabased ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 m) to produce lamella for cryogenic electron tomography.

cell biology↗

bioRxiv2022

Plasma FIB milling for the determination of structures in situ

Structural biology inside cells and tissues requires methods able to thin vitrified specimens to electron transparent thicknesses. Until now, focused ions beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit Gallium as an ion beam source. Here, we show that plasma-coupled ion sources can produce cryogenic lamella of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. In addition, these lamellae were produced in a prototype microscope equipped for long cryogenic run times (>1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate for the first time that plasma ion sources can be used for structural biology within cells, determining a structure in-situ to 4.9 [A] and describing a workflow upon which different plasmas can be examined to streamline lamella fabrication further.

biochemistry↗

bioRxiv2021

Cryptic SARS-CoV2-spike-with-sugar interactions revealed by 'universal' saturation transfer analysis

Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present universal saturation transfer analysis (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an end on manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and end on mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems. Extended AbstractThe surface proteins found on both pathogens and host cells mediate entry (and exit) and influence disease progression and transmission. Both types can bear host-generated post- translational modifications such as glycosylation that are essential for function but can confound biophysical methods used for dissecting key interactions. Several human viruses (including non- SARS-coronaviruses) attach to host cell-surface N-linked glycans that include forms of sialic acid (sialosides). There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we demonstrate quantitative extension of saturation transfer protein NMR methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The method couples objective resonance-identification via a deconvolution algorithm with Bloch-McConnell analysis to enable a structural, kinetic and thermodynamic analysis of ligand binding beyond previously-perceived limits of exchange rates, concentration or system. Using an automated and openly available workflow this universal saturation transfer analysis (uSTA) can be readily-applied in a range of even heavily-modified systems in a general manner to now obtain quantitative binding interaction parameters (KD, kEx). uSTA proved critical in mapping direct interactions between natural sialoside sugar ligands and relevant virus-surface attachment glycoproteins - SARS-CoV-2-spike and influenza-H1N1-haemagglutinin variants - by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile protein glycans (that also include sialosides). In B- origin-lineage-SARS-CoV-2 spike trimer end on-binding to sialoside sugars was revealed contrasting with extended surface-binding for heparin sugar ligands; uSTA-derived constraints used in structural modelling suggested sialoside-glycan binding sites in a beta-sheet-rich region of spike N-terminal domain (NTD). Consistent with this, uSTA-glycan binding was minimally- perturbed by antibodies that neutralize the ACE2-binding domain (RBD) but strongly disrupted in spike from the B1.1.7/alpha and B1.351/beta variants-of-concern, which possess hotspot mutations in the NTD. Sialoside binding in B-origin-lineage-NTD was unequivocally pinpointed by cryo-EM to a site that is created from residues that are notably deleted in variants (e.g. H69,V70,Y145 in alpha). An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a model in which this site in the NTD of B-origin-lineage-SARS-CoV-2 (but not in alpha/beta-variants) may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Together these confirm a novel binding mode mediated by the unusual NTD of SARS-CoV-2 and suggest how it may drive virulence and/or zoonosis via modulation of glycan attachment. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide ready, quantitative, widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.

biophysics↗