Biology subjects

Oorschot, V.

Publications and source records attributed to Oorschot, V..

12 recordsLinked to original sources

bioRxiv2026

Selective autophagy of whole micronuclei suppresses chromosomal instability

Chromosomal instability (CIN) arising from mitotic errors generates pervasive structural and numerical chromosome alterations fueling cancer evolution1-3. Entrapment of missegregated chromosomes within micronuclei exacerbates CIN by fostering repeated rounds of aberrant mitotic segregation4-6 and, following micronucleus rupture, promoting catastrophic chromosomal rearrangement processes such as chromothripsis5-9. Whether cellular mechanisms exist to restrain micronucleus-driven CIN has remained unclear. Developing a live-cell chromatin acidification sensor, we tracked micronuclei from genesis through subsequent cell cycles and observed frequent whole-micronucleus capture and acidification via the autophagy pathway. Autophagic targeting is selective for micronuclei with nuclear envelope defects seeded at mitotic exit. Our data indicate that these defects drive progressive loss of chromatin-nuclear envelope tethering, producing a mechanically altered state that is recognised by the autophagic machinery. Autophagy and rupture represent distinct micronuclear fates with opposing genomic consequences. Single-cell sequencing of fate-matched cells demonstrates complete digestion of the chromosomal contents of autophagy-targeted micronuclei, a process we term chromophagy (chromosome-autophagy). By eliminating micronuclei, chromophagy promotes chromosomal loss and arrests the intergenerational transmission of missegregated chromosomal material. This constrains the mutational consequences of micronucleation and suppresses micronucleus-mediated CIN.

cell biology↗

bioRxiv2026

Microtubules sustain the fidelity of cellularization in a coenocytic relative of animals

Cellularization is the coordinated division of a multinucleate cytoplasm into many cells.1-3 Multinucleation is a common life cycle strategy observed across eukaryotic lineages, including in microbial eukaryotes, fungi, plants and animals, and is associated with the ability to transition to a unicellular state through cellularization.4 In the best-studied model for this process, Drosophila melanogaster, cellularization requires the coordinated action of actin and microtubule (MT) networks to bring about the synchronous invagination of plasma membrane furrows, but the extent of conservation of these mechanisms across eukaryotes remains unknown.1,5,6 Here we investigate cellularization in the ichthyosporean Sphaeroforma arctica, a close relative of animals with a multinucleate life cycle stage.7-9 Using live cell imaging, ultrastructure expansion microscopy (U-ExM) and volume electron microscopy, we define the membrane, MT and actin dynamics that accompany cellularization in S. arctica. Using pharmacological inhibitors and centrifugation, we show that MTs, in addition to positioning nuclei, play a role in guiding nascent furrows to sustain equi-partitioning of nuclei and cytoplasm between daughter cells. Our findings indicate that cellularization is regulated through crosstalk between actin and MT networks, exhibiting mechanistic parallels with canonical cytokinesis, and establish S. arctica as a valuable model for investigating general principles of cellularization.

cell biology↗

bioRxiv2024

Sprint interval exercise disrupts mitochondrial ultrastructure driving a unique mitochondrial stress response and remodelling in humans

Exercise remains the most effective lifestyle intervention to remodel the mitochondrial network and to prevent most non-communicable diseases. Despite this, the molecular mechanisms by which different exercise prescriptions dictate mitochondrial remodelling are poorly understood in humans. Here, we show that, compared to moderate-intensity continuous exercise (MICE), sprint-interval exercise (SIE) - a known time-efficient high-intensity exercise - leads to mitochondrial stress and activates the mitochondrial unfolded protein response (UPRmt). The SIE-specific signature is characterized by a morphological and ultrastructural mitochondrial disturbance, concurrent with the activation of the integrated stress response (ISR) and mitochondrial quality control (MQC) pathways. When the respective exercises are repeated over time (8 weeks), our results demonstrate that moderate-intensity continuous training (MICT) and sprint-interval training (SIT) lead to a divergent mitochondrial remodelling. MICT elicits a mitochondrial adaptation characterized by an increase in markers of mitochondrial content, complex I activity, and enrichment of proteins involved in tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) system. On the other hand, SIT leads to proteomic enrichment of pathways involved in mitochondrial 1-Carbon metabolism and protein quality control, concurrently with improvements in mitochondrial respiratory function. Lastly, we have identified COX7A2L as a divergently regulated protein across groups, significantly accumulating in III2+IV1 respiratory supercomplexes only following SIT. In conclusion, our study provides mechanistic insights on how SIE and MICE divergently impact the post-exercise mitochondrial signalling, and subsequent long-term mitochondrial remodelling following training. These findings provide a strong basis for targeted exercise prescription to modulate specific mitochondrial adaptations in human skeletal muscle.

molecular biology↗

bioRxiv2023

Basal spot junctions of epithelial tissues respond to morphogenetic forces and regulate Hippo signalling

Organ size is controlled by numerous factors including mechanical forces, which are mediated in part by the Hippo pathway. In growing Drosophila melanogaster epithelial tissues, cytoskeletal tension influences Hippo signalling by modulating the subcellular localisation of key pathway proteins in different apical domains, namely adherens junctions, the sub-apical region and the medial apical cortex. Here, using both electron and light microscopy, we have discovered the existence of basal spot junctions in D. melanogaster epithelial tissues, and that they respond to morphogenetic forces and also influence Hippo signalling. Like adherens junctions, the Warts kinase is recruited to basal spot junctions via Ajuba and E-cadherin, which prevent Warts activation by segregating it from upstream Hippo pathway proteins. Basal spot junctions are prominent when tissues undergo morphogenesis and are highly sensitive to fluctuations in cytoskeletal tension. Basal spot junctions are distinct from focal adhesions, but the latter profoundly influences the abundance of spot junctions by modulating the basal-medial actomyosin network and tension experienced by spot junctions. Thus, basal spot junctions potentially couple morphogenetic forces to Hippo pathway activity and organ growth.

cell biology↗

bioRxiv2023

Dextromethorphan inhibits collagen transport in the endoplasmic reticulum eliciting an anti-fibrotic response in ex-vivo and in vitro models of pulmonary fibrosis

Excessive deposition of fibrillar collagen in the interstitial extracellular matrix (ECM) of human lung tissue causes fibrosis, which can ultimately lead to organ failure. Despite our understanding of the molecular mechanisms underlying the disease, a cure for pulmonary fibrosis has not yet been found. In this study, we screened an FDA-approved drug library containing 712 drugs and found that Dextromethorphan (DXM), a cough expectorant, significantly reduces the amount of excess fibrillar collagen deposited in the ECM in in-vitro cultured primary human lung fibroblasts (NHLF) and ex-vivo cultured human precision-cut lung slice (hPCLS) models of lung fibrosis. Reduced extracellular fibrillar collagen levels in the ECM upon DXM treatment are due to a reversible trafficking inhibition of collagen type I (COL1) in the endoplasmic reticulum (ER) in TANGO1 and HSP47 positive structures. Mass spectrometric analysis shows that DXM causes hyper-hydroxylation of proline and lysine residues on Collagen (COL1, COL3, COL4, COL5, COL7, COL12) and Latent-transforming growth factor beta-binding protein (LTBP1 and LTBP2) peptides coinciding with their secretion block. In addition, thermal proteome profiling of cells treated with DXM shows increased thermal stability of prolyl-hydroxylases such as P3H2, P3H3, P3H4, P4HA1 and P4HA2, suggesting a change in activity. Transcriptome analysis of pro-fibrotic stimulated NHLFs and hPCLS upon DXM treatment showed activation of an anti-fibrotic program via regulation of pathways such as those involved in the MMP-ADAMTS axis, WNT, and fibroblast-to-myofibroblast differentiation. Taken together, the data obtained from both in-vitro and ex-vivo models of fibrogenesis show that Dextromethorphan has potent anti-fibrotic activity by efficient inhibition of COL1 membrane trafficking in the ER.

cell biology↗

bioRxiv2023

Targeted volume Correlative Light and Electron Microscopy of an environmental marine microorganism

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods, along with the complexity and heterogeneity of environmental samples, pose great technical challenges. In the present study, we used a workflow based on a specific EM sample preparation, compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate with quantitative characterization of multiple organelles. We could show that this cell contains a single convoluted chloroplast and the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed chromatin features that could shed light on how transcriptional activity takes place in organisms where chromosomes are permanently condensed. Together with providing insights in dinoflagellates biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

cell biology↗

bioRxiv2022

Single-fibre proteomics reveals a greater fibre-type specific mitochondrial remodelling with moderate than high-intensity training.

Analyses of mitochondrial adaptations in human skeletal muscle have mostly used whole-muscle samples, where results may be confounded by the presence of a mixture of type I and II muscle fibres. Using our adapted mass spectrometry-based proteomics workflow, we provide new insights into fibre-specific mitochondrial differences in human skeletal muscle before and after training. Our findings challenge previous conclusions regarding the extent of fibre-type-specific remodelling of the mitochondrial proteome and highlight that most baseline differences in mitochondrial protein abundances between fibre types reported by us, and others, might be due to differences in total mitochondrial content or a consequence of adaptations to habitual physical activity (or inactivity). Most training-induced changes in different mitochondrial functional groups, in both fibre types, were stoichiometrically linked to changes in markers of mitochondrial content.

physiology↗

bioRxiv2021

Bimodal Endocytic Probe for Three-Dimensional Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) can infer molecular, functional and dynamic information to ultrastructure by linking information of different imaging modalities. One of the main challenges, especially in 3D-CLEM, is the accurate registration of fluorescent signals to electron microscopy (EM). Here, we present fluorescent BSA-gold (fBSA-Au), a bimodal endocytic tracer as fiducial marker for 2D and 3D CLEM applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent bovine serum albumin (BSA). The conjugate is efficiently endocytosed and distributed throughout the 3D endo-lysosomal network of the cells, and has an excellent visibility both in fluorescence microscopy (FM) and EM. We demonstrate the use of fBSA-Au in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryo-EM. As a fiducial marker, fBSA-Au facilitates rapid registration of regions of interest between FM and EM modalities and enables accurate (50-150 nm) correlation of fluorescence to EM data. Endocytosed fBSA-Au benefits from a homogenous 3D distribution throughout the endosomal system within the cell, and does not obscure any cellular ultrastructure. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo-)CLEM applications.

cell biology↗

bioRxiv2020

Macrophages provide a transient muscle stem cell niche via NAMPT secretion

Skeletal muscle is paradigmatic of a regenerative tissue that repairs itself via the activation of a resident stem cell1. Termed the satellite cell, these normally quiescent cells are induced to proliferate by ill-defined wound-derived signals2. Identifying the source and nature of these pro-regenerative cues has been hampered by an inability to visualise the complex cellular interactions that occur within the wound environment. We therefore developed a zebrafish muscle injury model to systematically capture satellite cell interactions within the injury site, in real time, throughout the repair process. This analysis identified that a specific subset of macrophages dwells within the injury, establishing a transient but obligate stem cell niche required for stem cell proliferation. Single cell profiling identified specific signals secreted from dwelling macrophages that include the cytokine, Nicotinamide phosphoribosyltransferase (NAMPT/Visfatin/PBEF). Here we show that NAMPT secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (CCR5) expressed on muscle stem cells. This analysis reveals that along with their well-described ability to modulate the pro-inflammatory and anti-inflammatory phases of wound repair, specific macrophage populations also provide a transient stem cell-activating niche, directly supplying pro-proliferative cues that govern the timing and rate of muscle stem cell-mediated repair processes.

cell biology↗

bioRxiv2019

Myelin and nodal plasticity modulate action potential conduction in the adult mouse brain

Central nervous system myelination increases action potential conduction velocity, however, it is unclear how myelination is coordinated to ensure the temporally precise arrival of action potentials, and facilitate information processing within cortical and associative circuits. Here, we show that mature myelin remains plastic in the adult mouse brain and can undergo subtle structural modifications to influence action potential arrival times. Repetitive transcranial magnetic stimulation and spatial learning, two stimuli that modify neuronal activity, alter the length of the nodes of Ranvier and the size of the periaxonal space within active brain regions. This change in the axon-glial configuration is independent of oligodendrogenesis and tunes conduction velocity to increase the synchronicity of action potential transit.

neuroscience↗

bioRxiv2019

Metformin rescues muscle function in BAG3 myofibrillar myopathy models

Dominant de novo mutations in the co-chaperone BAG3 cause a severe form of myofibrillar myopathy, exhibiting progressive muscle weakness, muscle structural failure, and protein aggregation.\n\nTo identify therapies we generated two zebrafish models, one conditionally expressing BAG3P209L and one with a nonsense mutation in bag3. Whilst transgenic BAG3P209L expressing fish display protein aggregation, modelling the early phase of the disease, bag3-/- fish demonstrate impaired autophagic activity, exercise dependent fibre disintegration, and reduced swimming activity, consistent with later stages.\n\nWe confirmed the presence of impaired autophagy in patient samples and screened autophagy promoting compounds for their effectiveness at removing protein aggregates, identifying nine including Metformin. Further evaluation demonstrated Metformin is not only able to remove the protein aggregates in zebrafish and human myoblasts but is also able to rescue the fibre disintegration and swimming deficit observed in the bag3-/- fish. Therefore, repurposing Metformin provides a promising therapy for BAG3 myopathy.

genetics↗

bioRxiv2018

Oxygen plasma focused ion beam scanning electron microscopy for biological samples

Over the past decade, gallium Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) has been established as a key technology for cellular tomography. The utility of this approach, however, is severely limited both by throughput and the limited selection of compatible sample preparation protocols. Here, we address these limitations and present oxygen plasma FIB (O-PFIB) as a new and versatile tool for cellular FIB-SEM tomography. Oxygen displays superior resin compatibility to other ion beams and produces curtain-free surfaces with minimal polishing. Our novel approach permits more flexible sample preparation and 30% faster data collection when compared to using gallium ion sources. We demonstrate this alternative FIB is applicable to a variety of embedding procedures and biological samples including brain tissue and whole organisms. Finally, we demonstrate the use of O-PFIB to produce targeted FIB-SEM tomograms through fiducial free en-block correlative light and electron microscopy.

cell biology↗