Biology subjects

Ronchi, P.

Publications and source records attributed to Ronchi, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Workflow and instrumentation for multiscale correlative imaging in voluminous cryogenic specimens

Cryo-electron tomography (cryo-ET) enables molecular-resolution imaging of intracellular environments, but is constrained by the need to generate electron-transparent sections of the biological specimen and thus a limited imaging volume. To alleviate such limitations, we present a multiscale correlative workflow that links cryo-confocal fluorescence microscopy (cryo-FLM), cryo-focused ion beam-scanning electron microscopy (cryo-FIB-SEM) volume imaging (cryo-vEM), and cryo-FIB serial lamellae lift-out for cryo-ET in high-pressure frozen voluminous specimens. An integral part of this workflow is a sample holder that preserves specimen orientation across instruments, reduces manual handling, and overcomes stage-tilt limitations in cryo-FIB-SEM hardware. Applied to vitrified colorectal cancer organoids, we demonstrate that this workflow enables fluorescence-guided site preparation for cryo-vEM, which provides biological context beyond the limited field of view of cryo-ET, and targeted lift-out for fabrication of multiple lamellae from defined regions of interest. The reconstructed tomograms reveal subcellular architecture and cell-cell interfaces. This workflow establishes a modular methodological framework for continuum-resolution cryogenic imaging of complex 3D biological specimens.

cell biology↗

bioRxiv2026

iSBEM: An Open-Source Workflow for Automated ROI Targeting in Volume Electron Microscopy

Serial Block Face - Scanning Electron Microscopy (SBF-SEM) is a volume EM method suited to investigate the 3D architecture of tissues and even entire organisms at high resolution. However, imaging large volumes in their entirety is time-consuming and not always necessary. Many research projects have a focused interest in well-defined sub-regions of the samples. The targeting and acquisition of such regions of interest (ROIs) are however currently conducted in a manual way and require heavy involvement of experienced operators. We present a workflow and an original open-source software tool (iSBEM), which allow automated targeting of ROIs in a large tissue sample, based on X-ray microscopy (XRM) maps. After an initial ROI identification and registration of the XRM map with the sample mounted on the SBF-SEM stage, iSBEM takes over the control of the microscope, triggering high resolution acquisitions at defined ROI positions, with minimal user intervention. We demonstrate the approach on two biologically distinct specimens -- malarial oocysts in infected mosquito midgut tissue, and immune cells in human kidney biopsies -- achieving significant improvement in acquisition throughput relative to manual operations, without compromising targeting precision. We also showcase the workflow in a correlative light-Xray-electron microscopy setup, which allowed us to further improve the correct target definition.

cell biology↗

bioRxiv2026

An Advanced Mobile Laboratory to enable field-based microbial ecology and cell biology across scales

Microbial biodiversity is central to ecosystem function, yet mechanistic insights into the cell biology of environmental organisms remain limited. The underlying challenges are twofold: most microbes remain uncultivable, and a persistent gap exists between field sampling and laboratory analyses. Here, we introduce the Advanced Mobile Laboratory (AML), a field-deployable platform that integrates confocal microscopy, image-enabled cell sorting, and cryo-preparation for expansion and electron microscopy. This setup enables immediate, standardized processing and analysis of environmental communities directly at the sampling site. We demonstrate its capability using marine eukaryotic plankton, showing how the AML enables multiscale investigations, from live imaging of natural communities to enabling ultrastructural and single-cell omics analyses, while minimizing sample degradation and enabling on-site experimentation. By bringing high-end sample preparation and analytical capacity into the field, the AML enables studying life in its natural context to mechanistically understand lifes diversity in the environment.

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↗

bioRxiv2025

The environmental stress response controls the biophysical properties of the cytoplasm and is critical for survival in quiescence

All organisms have evolved survival strategies to cope with changes in environmental conditions. Nutrient deprivation, one of the most frequently encountered stresses in nature, causes haploid budding yeast to enter a reversible state of non-proliferation known as quiescence, which entails extensive remodeling of gene expression, metabolism and the cellular biophysical properties. Yeast cells can adapt to and survive long periods of time in glucose starvation-induced quiescence, provided they are able to respire in the early stages of glucose withdrawal. When respiration is blocked during glucose withdrawal, cells prematurely age and exhibit markedly reduced survival and cytoplasmic diffusion. We find here that respiration is required to induce a quiescence-related gene expression program. Induction of this program prior to withdrawing glucose in respiration-inhibited cells bypasses the need for respiration and rescues survival and biophysical properties to levels seen in glucose-starved but respiration-competent cells. This rescue effect relies on proteomic adaptation, which partially occurs through inactivation of Ras/PKA signaling and activation of the environmental stress response via the transcription factors Msn2/4. This signaling cascade triggers the expression of stress response genes and modulates the cytoplasmic diffusion state of cells, ensuring long-term survival in quiescence even in the absence of respiration. Our results highlight the importance of stress adaptation in quiescence and aging, integrating gene expression control and modulation of cytoplasmic properties to maintain cell fitness.

cell biology↗

bioRxiv2025

Hydrophobic interactions of FG-nucleoporins are required for dilating nuclear membrane pores into selective transport channels after mitosis

Nuclear envelope (NE) reformation after mitosis is essential for daughter cell viability and requires tightly coordinated nuclear pore complex (NPC) assembly and nuclear membrane reformation. To reveal how these processes are mechanistically linked, we combined acute molecule perturbations in live cells with correlative 3D electron tomography or MINFLUX super-resolution microscopy. We show that degrading Nup62 during mitosis arrests NPC assembly at an intermediate step with smaller membrane pores and removes the whole central transport channel. Molecular dynamics simulations predicted that 32 copies of the central channel subcomplex, recruited into the previously unoccupied pore center, can self-associate via hydrophobic interactions to occupy the volume required for full pore size and exert an outward pushing force; indeed, disrupting these interactions during NPC assembly blocked pore dilation. Later in mitotic exit, perturbed cells exhibited impaired nuclear import, smaller nuclei, and looser NE spacing. Acute inhibition of nuclear import recapitulated these NE defects without affecting NPC assembly. Together, our findings reveal a new, two-step molecular mechanism linking NPC assembly and NE reformation. First, hydrophobic FG-nucleoporins dilate the assembling nuclear pore to its full width by forming the central transport channel, which then allows nuclear import-driven nuclear expansion leading to tight, regular NE membrane spacing.

cell biology↗

bioRxiv2025

Mesoglea biogenesis reveals a cryptic aboral valve for pressure regulation in cnidarian morphogenesis

Cnidarians are classically defined by a single oral opening, a hallmark of the "blind gut" model in early animal evolution. Here, we identify a pressure-sensitive aboral valve in Nematostella vectensis that operates independently of digestion. This valve dissipates elevated hydraulic pressure during morphogenesis, by expelling fluid through transient epidermal ruptures triggered by muscular ring opening. This unexpected function emerged from a comprehensive analysis of mesogleal basement membrane biogenesis. We show that the global dynamics of this extracellular matrix transduce muscular hydraulics to drive tissue rearrangement and stabilize shape, while localized FGFRb-dependent matrix remodeling establishes the aboral valve. By positioning the mesoglea as an integrator of biomechanics, tissue remodeling, and aboral valve function, these findings expand non-bilaterian openings beyond the digestive paradigm as a hydraulic regulator.

developmental biology↗

bioRxiv2024

Annulate Lamellae biogenesis is essential for nuclear pore function

Nuclear pore complexes (NPCs) enable nucleocytoplasmic transport. While NPCs primarily localize to the nuclear envelope (NE), they also appear in cytoplasmic endoplasmic reticulum (ER) membranes called annulate lamellae (AL). Though discovered in the mid-20th century, ALs function and biogenesis remain unclear. Previously considered exclusive to embryonic and malignant cells, we find AL in somatic mammalian cells. Under normal conditions, AL store pre-assembled NPCs (AL-NPCs) that integrate into the NE during G1 to support nuclear expansion. Upon pathological stimuli, AL transfer to the NE is impaired, leading to their cytoplasmic accumulation. RanBP2 (Nup358) is essential for AL biogenesis, with its phenylalanine-glycine (FG) repeats promoting AL-NPC scaffold oligomerization. ER-associated Climp63 (CKAP4) directs AL-NPCs to ER sheets and the NE. This AL-driven nuclear pore formation is complementary to the canonical routes, constituting a distinct NPC assembly pathway. Our work uncovers the biogenesis mechanism of AL and the nuclear function of this key cellular organelle.

cell biology↗

bioRxiv2024

Plasmodium falciparum disruption of pericyte angiopoietin-1 secretion contributes to barrier breakdown in a 3D brain microvessel model

Disruption of the vascular protective angiopoietin-Tie axis is common in cerebral malaria (CM) patients, who display elevated angiopoietin-2 (Ang-2) and reduced angiopoietin-1 (Ang-1) blood concentrations. The role of pericytes in CM pathogenesis remains unexplored, despite being a major source of brain Ang-1 secretion and evidence of pericyte damage observed in CM postmortem samples. Here we engineered a human 3D microfluidics-based brain microvessel model containing the minimal cellular components to replicate the angiopoietin-Tie axis, human primary brain microvascular endothelial cells and pericytes. This model replicated pericyte vessel coverage and ultrastructural interactions present in the brain microvasculature. When exposed to P. falciparum-iRBC egress products, 3D brain microvessels presented decreased Ang-1 secretion, increased vascular permeability, and minor ultrastructural changes in pericyte morphology. Notably, P. falciparum-mediated barrier disruption was partially reversed after pre-treatment with recombinant Ang-1 and the Tie-2 activator, AKB-9778. Our approach suggests a novel mechanistic role of pericytes in CM pathogenesis and highlights the potential of therapeutics that target the angiopoietin-Tie axis to rapidly counteract vascular dysfunction caused by P. falciparum. The paper explainedO_ST_ABSProblemC_ST_ABSCerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, resulting in the majority of [~]600000 malarial deaths annually. Despite anti-malarial drug administration upon hospitalization, fatality rates still range from 15-25% and many survivors suffer long term neurological disabilities. A common dysregulated vascular pathway identified in CM patients is the angiopoietin-Tie axis. Treatments that restore this vascular homeostatic pathway appear as a potential avenue for adjunctive therapies in experimental rodent CM models. Nevertheless, the use of rodent CM models for therapeutic discovery is not ideal, given that P. falciparum pathogenesis is species-specific. Therefore, the development of novel and advanced human 3D microvascular models offers new avenues to study disease pathogenesis and explore potential adjunctive CM treatments. ResultsIn this study, we generate a 3D human brain microvasculature model that reproduces in vivo interactions between two key cell types necessary to reproduce the protective angiopoietin-Tie axis: human brain endothelial cells and pericytes. Addition of P. falciparum-infected red blood cell (iRBC) egress products causes vascular disruption and hampers the release of the vascular protective factor, angiopoietin-1, from brain pericytes. 18-hour pre-treatment of Ang-1 for 18h prevents iRBC egress product-induced vascular disruption. A short pre-treatment of the microvessels with AKB-9778, a downstream pharmaceutical inducer of angiopoietin-Tie axis activity currently in phase II clinical trial for diabetic retinopathy, partially restores vascular integrity. Our study highlights the role of pericytes in CM and the therapeutic potential of interventions that restore the angiopoietin-Tie2 axis as adjunctive CM treatments. ImpactOur study demonstrates the potential of bioengineered vascular models to recapitulate dysregulated pathways previously characterized in malaria patients, and in providing a physiologically-relevant platform to test adjunctive therapies. The use of the 3D brain microvascular model has enhanced our understanding of the mechanisms behind CM pathogenesis, uncovering a previously unappreciated effect of P. falciparum on brain pericytes, linking angiopoietin-Tie axis dysregulation and microvasculature disruption. These findings pave the way for the identification of novel, fast-acting therapeutics, such as AKB-9778, to restore vascular integrity in CM patients.

microbiology↗

bioRxiv2024

In-cell structure and snapshots of copia retrotransposons in intact tissue by cryo-electron tomography

Long terminal repeat (LTR) retrotransposons belong to the transposable elements (TE), autonomously replicating genetic elements that integrate into the hosts genome. LTR retrotransposons represent a major component of genomes across the tree of life; some derived sequences have even been domesticated by the host to perform cellular functions in essential processes such as development. Among animals, Drosophila melanogaster serves as an important model organism for TE research, harboring several LTR retrotransposons, including the Ty1-copia family, which is evolutionarily related to retroviruses and forms virus-like particles (VLPs). The architectural organization of copia VLPs in situ has remained unknown. In this study, we use cryo-FIB milling and lift-out approaches to visualize copia VLPs in isolated ovarian cells and intact egg chambers and resolve the in situ copia capsid structure to 7.7 [A] resolution by cryo-ET. While cytosolic copia VLPs vary in size, nuclear VLPs are homogenous and form densely packed clusters, supporting a model in which nuclear import acts as a size selector. By analyzing flies deficient in the TE-suppressing PIWI-piRNA pathway, we observe a change in copia localization from cytosolic to nuclear during spermatogenesis in testes. Our findings provide insights into the cellular structural biology of an active LTR retrotransposon and shed light on the replication cycle of copia in the context of host gametogenesis.

biophysics↗

bioRxiv2023

Adhesion energy controls lipid binding-mediated endocytosis

Several bacterial toxins and viruses can deform membranes through multivalent binding to lipids for clathrin-independent endocytosis. However, it remains unclear, how membrane deformation and endocytic internalization are mechanistically linked. Here we show that many lipid-binding virions induce membrane deformation and clathrin-independent endocytosis, suggesting a common mechanism based on multivalent lipid binding by globular particles. We create a synthetic cellular system consisting of a lipid-anchored receptor in the form of GPI-anchored anti-GFP nanobodies and a multivalent globular binder exposing 180 regularly-spaced GFP molecules on its surface. We show that these globular, 40 nm diameter, particles bind to cells expressing the receptor, deform the plasma membrane upon adhesion and become endocytosed in a clathrin-independent manner. We explore the role of the membrane adhesion energy in endocytosis by using receptors with affinities varying over 7 orders of magnitude. Using this system, we find that once a threshold in adhesion energy is overcome to allow for membrane deformation, endocytosis occurs reliably. Multivalent, binding-induced membrane deformation by globular binders is thus sufficient for internalization to occur and we suggest it is the common, purely biophysical mechanism for lipid-binding mediated endocytosis of toxins and pathogens.

biophysics↗

bioRxiv2023

Life cycle-coupled evolution of mitosis in close relatives of animals

Eukaryotes have evolved towards one of two extremes along a spectrum of strategies for remodelling the nuclear envelope (NE) during cell division: disassembling the NE in an open mitosis or constructing an intranuclear spindle in a closed mitosis. Both classes of mitotic remodelling involve key differences in the core division machinery, but the evolutionary reasons for adopting a specific mechanism are unclear. Here, we use an integrated comparative genomics and ultrastructural imaging approach to investigate mitotic strategies in Ichthyosporea, close relatives of animals and fungi. We show that species within this clade have diverged towards either a fungal-like closed or an animal-like open mitosis, most likely to support distinct multi- or uninucleated states. Our results suggest that multinucleated life cycles favour the evolution of closed mitosis. One-Sentence SummaryMitotic specialization in animal relatives reveal that multinucleated life cycles favor the evolution of closed mitosis

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

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus

The first mitotic division of the initial cell is a key event in all multicellular organisms and is usually concomitant with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular and independent generations, the sporophyte and the gametophyte. Each generation deploys a distinct developmental program autonomously from an initial cell, whose first cell division sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first division of the initial cell and subsequently failure to produce basal structures (rhizoids and prostrate filaments) during both generations of the life cycle. Cloning-by-sequencing revealed that BAS encodes a type B" regulatory subunit of protein phosphatase 2A, and transcriptomic analysis of early developmental stages uncovered potential effector genes involved in setting up basal cell fate in this organism. The bas mutant phenotype is very similar to that observed in the distag (dis) mutants, which lack a functional TBCCd1 protein, at both the cellular and morphological levels. The high level of similarity of the dis and bas mutant phenotypes indicate that TBCCd1 and PP2A are two critical components of the cellular machinery that regulates the division of the initial cell and mediates the establishment of basal cell fate in the developing thallus.

developmental biology↗

bioRxiv2021

Identifying long-range synaptic inputs using genetically encoded labels and volume electron microscopy

Enzymes that facilitate the local deposition of electron dense reaction products have been widely used as labels in electron microscopy (EM). Peroxidases, in particular, can efficiently metabolize 3,3-diaminobenzidine tetrahydrochloride hydrate (DAB) to produce precipitates with high contrast under EM following heavy metal staining, and can be genetically encoded to facilitate the labeling of specific cell-types or organelles. Nevertheless, the peroxidase/DAB method has so far not been reported to work in combination with 3D volume EM techniques (e.g. Serial blockface electron microscopy, SBEM; Focused ion beam electron microscopy, FIBSEM) because the surfactant treatment needed for efficient reagent penetration disrupts tissue ultrastructure and because these methods require the deposition of large amounts of heavy metals that can obscure DAB precipitates. However, a recently described peroxidase with enhanced enzymatic activity (dAPEX2) appears to successfully deposit EM-visible DAB products in thick tissue without surfactant treatment. Here we demonstrate that multiplexed dAPEX2/DAB tagging is compatible with both FIBSEM and SBEM volume EM approaches and use them to map long-range genetically identified synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in the mouse brain.

neuroscience↗

bioRxiv2021

Integrated light and electron microscopy continuum resolution imaging of 3D cell cultures

3D cell cultures, in particular organoids, are emerging models to investigate healthy or diseased tissues. Understanding the complex cellular sociology in organoids requires integration of imaging modalities across spatial and temporal scales. We present a multi-scale imaging approach that traverses millimeter-scale live-cell light microscopy to nano-scale volume electron microscopy by performing 3D cell cultures in a single carrier amenable to all imaging steps. This allows to follow organoids growth, probe their morphology with fluorescent markers, identify areas of interest and analyze their 3D ultrastructure. We demonstrate this workflow on mouse and human 3D cultures, and use automated image segmentation to annotate and quantitatively analyze subcellular structures in patient-derived colorectal cancer organoids. Our analyses reveal local organization of diffraction-limited cell junctions in compact and polarized epithelia. The continuum resolution imaging pipeline is thus suited to foster basic and translational organoid research by simultaneously exploiting the advantages of light and electron microscopy. HighlightsO_LIEstablishment of 3D cell cultures in sample carriers directly amenable to high-pressure freezing (HPF) C_LIO_LI3D cell cultures in HPF carriers allow drug treatment and live-cell imaging C_LIO_LIMulti-scale imaging of 3D cultures from live-cell light microscopy to volume electron microscopy C_LIO_LIEstablishments of HPF conditions for mouse and patient-derived organoids C_LIO_LIDeep-learning automatic segmentation of ultrastructural detail and quantitative data-mining reveal different subcellular organization associated with epithelium polarity C_LI

cell biology↗

bioRxiv2021

Fluorescence-based 3D targeting of FIB-SEM acquisition of small volumes in large samples

Cells are three dimensional objects. Therefore, 3D electron microscopy is often crucial for correct interpretation of ultrastructural data. Today samples are frequently imaged in 3D at ultrastructural resolution using volume Scanning Electron Microscopy (SEM) methods such as Focused Ion Beam (FIB) SEM and Serial Block face SEM. While these imaging modalities allow for automated data acquisition, precise targeting of (small) volumes of interest within a large sample remains challenging. Here, we provide an easy and reliable approach to target FIB-SEM acquisition of fluorescently labelled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeting based on confocal acquisition of the fluorescence signal in the resin block. Targeted trimming of the block exposes the cell of interest and laser branding of the surface after trimming creates landmarks to precisely position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as a 3D culture of mouse primary mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before. SummaryRonchi et al. present a workflow to facilitate the precise targeting of three-dimensional (3D) Electron Microscopy acquisitions, guided by fluorescence. This method allows ultrastructural visualization of single cells within a millimeter-range large specimen, based on molecular identity characterized by fluorescence.

cell biology↗

bioRxiv2020

In cell architecture of the nuclear pore complex and snapshots of its turnover

Nuclear pore complexes (NPCs) mediate exchange across the nuclear envelope. They consist of hundreds of proteins called nucleoporins (Nups) that assemble in multiple copies to fuse the inner and outer nuclear membranes. Elucidating the molecular function and architecture of NPCs imposes a formidable challenge and requires the convergence of in vitro and in situ approaches. How exactly NPC architecture accommodates processes such as mRNA export or NPC assembly and turnover inside of cells remains poorly understood. Here we combine integrated in situ structural biology, correlative light and electron microscopy with yeast genetics to structurally analyze NPCs within the native context of Saccharomyces cerevisiae cells under conditions of starvation and exponential growth. We find an unanticipated in situ layout of nucleoporins with respect to overall dimensions and conformation of the NPC scaffold that could not have been predicted from previous in vitro analysis. Particularly striking is the configuration of the Nup159 complex, which appears critical to spatially accommodate not only mRNA export but also NPC turnover by selective autophagy. We capture structural snapshots of NPC turnover, revealing that it occurs through nuclear envelope herniae and NPC-containing nuclear vesicles. Our study provides the basis for understanding the various membrane remodeling events that happen at the interface of the nuclear envelope with the autophagy apparatus and emphasizes the need of investigating macromolecular complexes in their cellular context.

biophysics↗