Biology subjects

Domart, M.-C.

Publications and source records attributed to Domart, M.-C..

7 recordsLinked to original sources

bioRxiv2025

VP-CLEM-Kit: An accessible pipeline for visual proteomics using super resolution volume correlative light and electron microscopy (SR-vCLEM)

Visual proteomics (VP) aims to allow researchers to visualise, measure and analyse proteins in the context of cell and tissue structure in health and disease. VP is becoming a reality through technological advances across several domains, including in situ structural biology and correlative light and electron microscopy (CLEM). However, widespread adoption remains limited due to the complexity and cost of the various VP approaches reported to date. Here we present the VP-CLEM-Kit, a disruptive cost-effective pipeline for super resolution volume CLEM (SR-vCLEM) that can be implemented with minimal advanced electron microscopy expertise and equipment, making it accessible to light microscopy facilities and research labs. SR-vCLEM is based on in-resin fluorescence (IRF), where fluorophores are preserved through processing into resin. The easyIRF protocol reported here reduces the requirement for complex costly sample preparation equipment and toxic chemicals compared to standard IRF protocols. easyIRF blocks are cut into ultrathin sections that are imaged using tomoSTORM, a new modular and cost-effective openFrame-based light microscope controlled by the open-source software package Micro-Manager that provides serial single molecule localisation microscopy in array tomography format. Sections are then post-stained and imaged using a tabletop scanning electron microscope controlled by open-source SBEMimage software to run in array tomography format. We demonstrate the potential of the VP-CLEM-Kit by imaging organelle reporters in human cell lines and stem cell derived neurons, fluorescently labelled protein in neurons, and immunolabelled cells in human kidney biopsy tissue from transplant patients. The VP-CLEM-Kit delivers a [~]5-fold improvement in resolution at a [~]7-fold lower cost, and thus provides new technical capability as well as a blueprint for more equitable access to advanced imaging workflows.

cell biology↗

bioRxiv2024

A distinctive PI(4,5)P2 compartment forms during entosis and related engulfment processes

Entosis is a form of cell cannibalism prevalent in human tumours. During entosis, one live and viable epithelial cell is completely internalised by another, then housed inside a large, single-membrane, endolysosomal vacuole in the host cytosol. The composition and maturation of this specialised, macroendocytic compartment has not been fully defined, but ultimately, the inner cell is killed and digested by host lysosomes. Here, we investigate the molecular characteristics and maturation profile of the entotic vacuole. Like phagosomes and macropinosomes, this vacuole undergoes a series of phospholipid modifications, but its maturation profile bears distinctive dynamics. While PI(4,5)P2 is lost rapidly during phagosome maturation, entosis yields an unusual, intracellular PI(4,5)P2-positive compartment, that can persist for hours, suggesting vacuole maturation is uncoupled from membrane scission. Loss of PI(4,5)P2 is eventually triggered, a requisite step for lysosomal killing of the internalised cell. More broadly, PI(4,5)P2-positive vacuoles also form during T-cell engulfment by thymic nurse cells, dependent on ROCK activity, suggesting this distinctive compartment represents a shared feature of entosis-like cell engulfments.

cell biology↗

bioRxiv2023

CLEM-Reg: An automated point cloud based registration algorithm for correlative light and volume electron microscopy

Correlative light and volume electron microscopy (vCLEM) is a powerful imaging technique that enables the visualisation of fluorescently labelled proteins within their ultrastructural context on a subcellular level. Currently, expert microscopists align vCLEM acquisitions using time-consuming and subjective manual methods. This paper presents CLEM-Reg, an algorithm that automates the 3D alignment of vCLEM datasets by leveraging probabilistic point cloud registration techniques. These point clouds are derived from segmentations of common structures in each modality, created by state-of-the-art open-source methods, with the option to leverage alternative tools from other plugins or platforms. CLEM-Reg drastically reduces the time required to register vCLEM datasets to a few minutes and achieves correlation of fluorescent signal to sub-micron target structures in EM on three newly acquired vCLEM benchmark datasets (fluorescence microscopy combined with FIB-SEM or SBF-SEM). CLEM-Reg was then used to automatically obtain vCLEM overlays to unambiguously identify TGN46-positive transport carriers involved in the trafficking of proteins between the trans-Golgi network and plasma membrane. The datasets are available in the EMPIAR and BioStudies public image archives for reuse in testing and developing multimodal registration algorithms by the wider community. A napari plugin integrating the algorithm is also provided to aid end-user adoption.

cell biology↗

bioRxiv2022

NDR1/2 kinases regulate membrane trafficking, enable efficient autophagy and prevent neurodegeneration

Autophagy is essential for neuronal development and its deregulation contributes to neurodegenerative diseases. NDR1 and NDR2 are highly conserved kinases implicated in neuronal development, mitochondrial health and autophagy, but how they affect mammalian brain development in vivo is not known. Using single and double Ndr1/2 knockout mouse models we show that, dual, but not individual loss of Ndr1/2 in neurons causes neurodegeneration during brain development, but also in adult mice. Proteomic and phosphoproteomic comparisons between Ndr1/2 knockout and control brains revealed novel kinase substrates and indicated that endocytosis is significantly affected in the absence of NDR1/2. We validated the endocytic protein, Raph1/Lpd1 as a novel NDR1/2 substrate and showed that both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. In NDR1/2 knockout brains, we observed prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment of protein homeostasis. Furthermore, the levels of LC3-positive autophagosomes were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation and neurotoxicity. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health. HighlightsO_LIDual neuronal Ndr1 and Ndr2 knockout during development or in adult mice causes neurodegeneration. C_LIO_LIPhosphoproteomics comparison of Ndr1/2 knockouts with control littermates shows endocytosis and membrane trafficking to be affected and reveals novel substrates. C_LIO_LIRaph1/Lamellipodin is a novel NDR1/2 substrate that is required for TfR endocytosis. C_LIO_LINdr1/2 knockout brains exhibit a severe defect in ubiquitinated protein clearance and reduced autophagy. C_LIO_LINDR1/2 and Raph1 are required for the trafficking of the only transmembrane autophagy protein, ATG9A. C_LI

neuroscience↗

bioRxiv2021

LAP1 regulates nuclear plasticity to enable constrained migration

Metastasis involves dissemination of cancer cells away from a primary tumour and colonisation at distal sites. During this process, cancer cells must negotiate multiple physical constraints imposed by the microenvironment and tissue structure. The biophysical properties of the nucleus must be tuned since they pose a challenge to constrained migration. By analysing nuclear genes upregulated during the acquisition of metastatic potential, we discovered increased expression of the inner nuclear membrane protein LAP1 in metastatic melanoma cells and at the invasive fronts of human primary tumours and in metastases. Human cells express two LAP1 isoforms (LAP1B and LAP1C), which differ in their amino terminus. We found that whereas the longer isoform, LAP1B, binds more strongly to nuclear lamins and has restricted motility within the nuclear envelope, the shorter isoform, LAP1C, favours nuclear envelope blebbing and allows migration through constraints. We propose that LAP1 renders the nucleus plastic and enhances melanoma aggressiveness.

cell biology↗

bioRxiv2019

In vivo control of Toxoplasma gondii by zebrafish macrophages

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we develop a zebrafish infection model to study the innate immune response to Toxoplasma in vivo. By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision driven automated image analysis, we reveal that Toxoplasma invades and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are being cleared in vivo, we analyzed Toxoplasma-macrophage interactions using time-lapse and correlative light and electron microscopy. Strikingly, macrophages are recruited to the infection site and play a key role in Toxoplasma control. These results highlight in vivo control of Toxoplasma by macrophages, and illuminate the possibility to exploit zebrafish for discoveries within the field of parasite immunity.

microbiology↗

bioRxiv2019

Differential spatiotemporal targeting of Toxoplasma and Salmonella by GBP1 assembles caspase signalling platforms

Guanylate binding proteins (GBPs), a family of interferon (IFN)-inducible GTPases, can promote cell-intrinsic defense by removal of intracellular microbial replicative niches through host cell death. GBPs target pathogen-containing vacuoles or the pathogen itself, and assist in membrane-disruption and release of microbial molecules that trigger cell death by activating the inflammasomes. We previously showed that GBP1 mediates atypical apoptosis or pyroptosis of human macrophages infected with Toxoplasma gondii (Tg) or Salmonella enterica Typhimurium (STm), respectively. In mice, the p47 Immunity-related GTPases (IRGs) control the recruitment of GBPs to microbe-containing vacuoles and subsequent cell death. However, humans are devoid of functional IRGs, and the pathogen-proximal immune detection mechanisms by GBP1 are poorly understood. Here, we describe two novel single-cell assays which show that GBP1 promotes the lysis of Tg-containing vacuoles and Tg plasma membrane, resulting in the cytosolic detection of Tg-DNA. In contrast, we show GBP1 only targets cytosolic STm and does not contribute to bacterial escape into the cytosol of human macrophages. GBP1 interacts with caspase-4 and recruits it directly to the bacterial surface, where caspase-4 can be activated by LPS. During STm infection, caspase-1 cleaves and inactivates GBP1 at Asp192, a site conserved in related mammalian GBP1 proteins but not in murine Gbps. STm-infected human macrophages expressing a cleavage-deficient GBP1 mutant exhibit higher pyroptosis due to the absence of caspase-1-mediated feedback inhibition of the GBP1-caspase-4 pathway. Our comparative studies elucidate microbe-specific spatiotemporal roles of GBP1 in detecting infection and the assembly and regulation of divergent caspase signaling platforms.

microbiology↗