Biology subjects

Grange, M.

Publications and source records attributed to Grange, M..

At least 19 recordsLinked to original sources

bioRxiv2026

CD44-CD206 axis drives dendritic cell loss and immunosuppression in immaturely glycosylated bladder tumours

Antigen-presenting-cell dysfunction drives immune exclusion, but how tumour glycans disable these cells remains incompletely defined. Immature O-glycosylation, marked by Tn and sialyl-Tn antigens, drives myeloid dysfunction in bladder cancer. Transcriptomics and human tumour profiling linked this state to reduced representation of antigen-presenting myeloid cells and attenuated lymphoid-effector programmes. In glycoengineered models, immature O-glycans promoted immunoregulatory macrophage phenotypes and contact-dependent death of monocyte-derived dendritic cells (moDCs), while surviving cells showed impaired maturation and T-cell activation. Tumour-cell CD44 standard isoform was required for DC death, which was accompanied by inflammasome-associated proteins and gasdermin D cleavage, consistent with pyroptosis. Receptor-discovery approaches converged on CD206, which bound immaturely glycosylated CD44. CD206 blockade restored DC viability. In immunocompetent mice, immature glycosylation accelerated tumour growth and depleted conventional DCs, whereas CD206 blockade restrained tumour progression and restored local and nodal myeloid and lymphoid immunity. These findings identify CD44-CD206 as a targetable glycoimmune checkpoint that promotes immune exclusion.

immunology↗

bioRxiv2026

Femtosecond pulsed laser ablation for in-tissue electron cryo-tomography

In situ electron cryo-tomography (cryo-ET) is a powerful tool for visualising biology inside cells at the molecular scale. The routine application of cryo-ET in tissue samples promises biological insight beyond single cells, but is currently limited by the technical challenge of preparing high quality, electron-transparent specimens from thick (>100 m) biopsies efficiently. Here, we demonstrate that cryogenic femtosecond pulsed laser ablation (cryo-fs-PLA) can accelerate the preparation of vitreous specimens of mammalian brain tissue for cryo-ET. Using cryogenic fluorescence microscopy, regions of interest within high-pressure frozen tissue can be specifically targeted, and cryo-fs-PLA can be employed as a preparative step for serial cryogenic lift-out experiments. We define cryo-fs-PLA parameters and determine ablation rates for two different laser wavelengths, and demonstrate that tilt series collected on lamellae fabricated from sites exposed to cryo-fs-PLA largely maintain their vitrification state. Incorporating cryo-fs-PLA into existing cryo-ET workflows has the potential to unlock the sample preparation of tissues at the throughput necessary for routine in situ cell biology of multicellular samples.

biochemistry↗

bioRxiv2026

Genome Organizer SATB1 selectively activates a defined subset of EMT genes driving metastatic breast cancer

SATB1 reshapes chromatin architecture and transcriptional programs to promote breast cancer metastasis. However, its key downstream effectors remain incompletely defined. Here, we aimed to identify actionable drivers of invasion by focusing on epithelial-mesenchymal transition (EMT) genes. We identified 98 of 300 curated EMT-promoting genes as direct SATB1 targets in human breast epithelial cells (MCF10A-1) rendered tumorigenic with metastatic traits by SATB1 transduction, using Global Run-On Sequencing (GRO-seq) to measure nascent transcripts. These SATB1-activated EMT genes regulate extracellular matrix remodeling, hypoxia-responsive transcriptional programs, and tumor microenvironmental programs linking angiogenesis and immune evasion, collectively enhancing metastatic competence. Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by frequent metastasis and chemoresistance. Among the four TNBC molecular subtypes, the 98 SATB1-regulated EMT genes were significantly enriched and activated in the Basal-like 2 (BL2) subtype (Fishers exact test: p = 4.53e-9), which is associated with aggressive behavior, poorer clinical outcomes, and reduced treatment responsiveness. In contrast, SATB1-independent EMT genes showed no enrichment in BL2, indicating selective regulation of EMT genes by SATB1. We further analyzed nascent transcripts induced by the environmental carcinogen benzo[a]pyrene (B[a]P), a known breast carcinogen. Half of the 72 EMT genes activated after short-term B[a]P exposure overlapped with SATB1-dependent EMT genes, indicating that two distinct etiologies, SATB1 and B[a]P, converge on a largely shared network of invasion-promoting genes. These results show that EMT genes are not globally or randomly activated in breast cancer but are selectively activated, defining an EMT gene network associated with metastatic risk. This gene signature may serve as a prognostic marker pending further validation.

cancer biology↗

bioRxiv2026

Interleaved multi-magnification cryo-electron tomography bridges cellular and structural biology

Cryo-electron tomography (cryo-ET) enables in situ structural analysis of macromolecular complexes within native cellular environments. However, the limited field of view required for high-resolution structure determination necessarily restricts a wider assessment of the broader cellular context. We present a multi-magnification cryo-ET acquisition strategy that integrates low- and high-magnification information from coincident sample regions during the same tilt-series. By interleaving acquisition of the magnifications at each tilt angle, this strategy enables simultaneous collection of large field-of-view, low-resolution tomograms and high-resolution, small field-of-view tomograms while minimising the impact of the increased electron dose. We demonstrate that we can capture cellular organisation across tens of microns, while still enabling subtomogram averaging to resolutions below 4 [A]. This integrated acquisition framework establishes a practical route to multi-scale cryo-ET, bridging molecular and cellular scales for more comprehensive biological insight.

biochemistry↗

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↗

bioRxiv2024

Charting the molecular landscape of neuronal organisation within the hippocampus using cryo electron tomography

Cellular cryo-electron tomography (cryoET) enables the capture of detailed structural information within a biologically relevant environment. However, information in more complex samples, such as tissues, is lacking. Importantly, these observations need to be set in context of populations; imaging on the molecular scale to-date is limited to few observations in-situ that struggle to be generalised. This is due to limitations in throughput and versatility employed by current instrumentation. Here, we utilise plasma focused ion beam milling to examine the molecular landscape of mouse hippocampus by cryoET in targeted regions across multiple individuals revealing the complex organisation of macromolecules from the CA1 strata pyramidale (sp) to radiatum (sr). Our data represent a molecular atlas, producing snapshots of hippocampal architecture in adult mouse. The combination of instrumentation and application of technical advancements provides a framework to explore specific structural questions within native tissues in a targeted manner.

biochemistry↗

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↗

bioRxiv2023

Antiviral activity of intracellular nanobodies targeting the influenza virus RNA-polymerase core

Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol). FluPol displays structural flexibility related to distinct functional states, from an inactive form to conformations competent for replication and transcription. FluPol machinery is constituted by a structurally-invariant core comprising the PB1 subunit stabilized with PA and PB2 domains, whereas the PA endonuclease and PB2 C-domains can pack in different configurations around the core. To get insights into the functioning of FluPol, we selected single-domain nanobodies (VHHs) specific of the influenza A FluPol core. When expressed intracellularly, several of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) targets PA, but preferentially bind the PA-PB1 dimer with an affinity below the nanomolar range. Ectopic intracellular expression of VHH16 in virus permissive cells blocks multiplication of different influenza A subtypes, even when induced at late times post-infection. VHH16 was found to impair the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin {beta} RanBP5 and subsequent steps in FluPol assembly. These data suggest that the VHH16 neutralization activity is likely due to an alteration of the import of the PA-PB1 dimer into the nucleus, resulting to an inhibition of FluPol functioning. VHH16 binding site represent a potential target for antiviral development. Author SummaryThe influenza virus RNA-polymerase (FluPol) ensures genome transcription and replication in the nucleus of the infected cells. To select ligands able to block FluPol activities, we screened a library of phages encoding nanobodies and resulting from the immunization of a llama with FluPol subunits. When expressed intracellularly, one of the nanobodies displays highly efficient FluPol blocking and virus neutralizing activities. This nanobody binds FluPol with high affinity and recognizes preferentially the PA-PB1 assembled subunits. Furthermore, it was found to interfere with the transport of the PA-PB1 dimer into the nucleus, suggesting that targeting FluPol trafficking between the cytoplasm and the nucleus may constitute a powerful strategy to develop new antivirals.

microbiology↗

bioRxiv2023

NECing goes: flexibility of the herpesvirus nuclear egress complex

The nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without breaking the nuclear envelope barrier. It assembles into a lattice on the inner nuclear membrane enveloping newly assembled nucleocapsids, which bud into the perinuclear space. The primary virion envelope subsequently fuses with the outer nuclear membrane, releasing the capsid into the cytosol. Here we interrogated the NEC in the context of intact cells infected with pseudorabies or herpes simplex virus using focused-ion beam milling and electron cryo- tomography. We determined the structure of NEC in different conformations and show that it consists of a flexible hexameric lattice that generates curvature through a combination of ordered and disordered domains. After interrogating the intermediate stages of capsid formation, we show that capsid vertex binding may initiate envelopment but does not directly induce curvature formation. These data and many examples of the intermediate stages of nuclear egress paint a detailed holistic view of a versatile transport system.

microbiology↗

bioRxiv2023

In situ structures from relaxed cardiac myofibrils reveal the organization of the muscle thick filament

The thick filament is a key component of sarcomeres, the basic force-generating and load-bearing unit of striated muscle1. Mutations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2, 3. Despite this central importance for sarcomere force generation, it remains unclear how thick filaments are structurally organized and how its components interact with each other and with thin filaments to enable highly regulated muscle contraction. Here, we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by electron cryo-tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin heads and tails, myosin-binding protein C (MyBP-C) and titin, elucidating the structural basis for their interaction during muscle contraction. The arrangement of myosin heads is variable depending on their position along the filament, suggesting that they have different capacities in terms of strain susceptibility and activation. Myosin tails exhibit a distinct arrangement and pattern of interactions. These are likely orchestrated by three alpha and three beta titin chains that are arranged like a spring, suggesting the existence of specialized roles of thick filament segments in length-dependent activation and contraction. Surprisingly, while the three titin alpha chains run along the entire length of the thick filament, titin beta does not. The structure also demonstrates that the C-terminal region of MyBP-C binds myosin tails and unexpectedly also directly interacts with the myosin heads, suggesting a previously undescribed direct role in the preservation of the myosin OFF state. Furthermore, we visualize how MyBP-C forms links between thin and thick filaments. These findings establish a robust groundwork for forthcoming research endeavors aiming to explore muscle disorders that involve sarcomeric structural components.

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

Deeply hidden genome organization directly mediated by SATB1

Mammalian genomes are organized by multi-level folding, yet how this organization contributes to cell type-specific transcription remain unclear. A nuclear protein SATB1 forms a SATB1-rich subnuclear structure that resists high-salt extraction. SATB1 binds in the minor groove of double-stranded base-unpairing regions (BURs), genomic elements with high unwinding propensities. We uncovered that SATB1 establishes a two-tiered chromatin organization, one through indirect binding and another by direct binding of BURs. Published ChIP-seq datasets show SATB1 binding to highly accessible chromatin at enhancers and CTCF sites, but not to BURs. By employing urea ChIP-seq, which retains only directly bound protein:DNA complexes, we found that BURs, but not CTCF sites, are direct SATB1 binding targets genome-wide. BURs bound to the SATB1 nuclear substructure interact with accessible chromatin crossing multiple topologically associated domains (TADs). SATB1 is required for these megabase-scale interactions linked to cell type-specific gene expression. BURs are highly enriched within lamina associated domains (LADs), but some ([~]10%) are found in gene-rich accessible chromatin outside LADs as well. Only a subset of BURs is bound to SATB1 depending on cell type. Notably, despite the mutually exclusive SATB1-binding profiles uncovered by the two ChIP-seq methods, we found most peaks in both profiles are valid and require SATB1. Based on these and previous data, we propose that the SATB1 protein network forms a chromatin scaffold, providing an interface that connects accessible chromatin to a subnuclear architectural structure, thereby facilitating the three-dimensional organization linked to cell type-specific gene expression.

genomics↗

bioRxiv2021

Structure of the thin filament in native skeletal muscles reveals its interaction with nebulin and two distinct conformations of myosin

Nebulin is a major structural protein of skeletal sarcomeres and is essential for proper assembly and contraction of skeletal muscle1. It stabilises and regulates the length of thin filaments,2 but the structural mechanism remains nebulous. Using electron cryotomography and sub-tomogram averaging, we present the first structure of native nebulin bound to thin filaments within the A-band and I-band of intact sarcomeres. This in-situ reconstruction reveals unprecedented detail of interaction at pseudo-atomic resolution between nebulin and actin, providing the basis for understanding the structural and regulatory roles of nebulin. The position of nebulin on the thin filament indicates that there is no contact to tropomyosin or myosin, but an unexpected interaction with a troponin-T linker, possibly through two binding motifs on nebulin. In addition, our structure of myosin bound to the thin filaments reveals different conformations of the neck domain, both within the same sarcomere and when compared to purified structures, highlighting an inherent structural variability in muscle. We provide a complete description of cross-bridge formation on fully native, nebulin-containing thin filaments at near-atomic scale. Our structures establish the molecular basis for the role of nebulin as a thin filament "molecular ruler" and the impact of nemaline myopathies mutations that will aid future development of therapies.

biochemistry↗

bioRxiv2020

Demographics and Employment of Max-Planck Society's Postdocs

The recently founded Max Planck PostdocNet brings together postdoctoral researchers (or postdocs) from the Max Planck Society (MPS), provides representation for the postdoctoral community across all Max Planck Institutes (MPI) and associated institutes, and advocates for their interests on their behalf. At the 2019 founding meeting, MPS postdocs quickly raised their concerns about their employment situation and their associated social and working conditions. Subsequently, the PostdocNet conducted the first survey targeting exclusively postdoctoral researchers to gather information on their demographics, employment situations and social conditions. This report presents the results of this survey, providing a thorough characterization of the postdoc demographics as well as the working conditions experienced by the postdoctoral community of the MPS. Remarkably, the survey analysis revealed a number of disparities in the access to employment type, wage level and social benefits. These results will guide future and present MPS postdoctoral researchers and their employers at the MPS to thrive for equality and fairness. Moreover, these results should be of help to the MPS to establish, improve and maintain optimal working conditions for MPS postdocs.

scientific communication and education↗