Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure

Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo

Super-resolution light microscopy, correlative light and electron microscopy, and volume electron microscopy are revolutionising the way in which biological samples are examined and understood. Here, we combine these approaches to deliver super-accurate correlation of fluorescent proteins to cellular structures. We show that YFP and GFP have enhanced blinking properties when embedded in acrylic resin and imaged under partial vacuum, enabling in vacuo single molecule localisation microscopy. In conventional section-based correlative microscopy experiments, the specimen must be moved between imaging systems and/or further manipulated for optimal viewing. These steps can introduce undesirable alterations in the specimen, and complicate correlation between imaging modalities. We avoided these issues by using a scanning electron microscope with integrated optical microscope to acquire both localisation and electron microscopy images, which could then be precisely correlated. Collecting data from ultrathin sections also improved the axial resolution and signal-to-noise ratio of the raw localisation microscopy data. Expanding data collection across an array of sections will allow 3-dimensional correlation over unprecedented volumes. The performance of this technique is demonstrated on vaccinia virus (with YFP) and diacylglycerol in cellular membranes (with GFP)

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

Human guanylate binding proteins (GBPs), a family of IFNγ-inducible GTPases, promote cell-intrinsic defence against pathogens and host cell death. We previously identified GBP1 as a mediator of cell death of human macrophages infected with Toxoplasma gondii (Tg) or Salmonella Typhimurium (STm). How GBP1 targets microbes for AIM2 activation during Tg infection and caspase-4 during STm infection remains unclear. Here, using correlative light and electron microscopy and EdU labelling of Tg-DNA, we reveal that GBP1-decorated parasitophorous vacuoles (PVs) lose membrane integrity and release Tg-DNA for detection by AIM2-ASC-CASP8. In contrast, differential staining of cytosolic and vacuolar STm revealed that GBP1 does not contribute to STm escape into the cytosol but decorates almost all cytosolic STm leading to the recruitment of caspase-4. Caspase-5, which can bind LPS and whose expression is upregulated by IFNγ, does not target STm pointing to a key role for caspase-4 in pyroptosis. We also uncover a regulatory mechanism involving the inactivation of GBP1 by its cleavage at Asp192 by caspase-1. Cells expressing non-cleavable GBP1D192E therefore undergo higher caspase-4-driven pyroptosis during STm infection. Taken together, our comparative studies elucidate microbe-specific spatiotemporal roles of GBP1 in inducing cell death by leading to assembly and regulation of divergent caspase signalling platforms

Mitosis can drive cell cannibalism through entosis.

Entosis is a form of epithelial cell cannibalism that is prevalent in human cancer, typically triggered by loss of matrix adhesion. Here, we report an alternative mechanism for entosis in human epithelial cells, driven by mitosis. Mitotic entosis is regulated by Cdc42, which controls mitotic morphology. Cdc42 depletion enhances mitotic deadhesion and rounding, and these biophysical changes, which depend on RhoA activation and are phenocopied by Rap1 inhibition, permit subsequent entosis. Mitotic entosis occurs constitutively in some human cancer cell lines and mitotic index correlates with cell cannibalism in primary human breast tumours. Adherent, wild-type cells can act efficiently as entotic hosts, suggesting that normal epithelia may engulf and kill aberrantly dividing neighbours. Finally, we report that Paclitaxel/taxol promotes mitotic rounding and subsequent entosis, revealing an unconventional activity of this drug. Together, our data uncover an intriguing link between cell division and cannibalism, of significance to both cancer and chemotherapy

Entosis Controls a Developmental Cell Clearance in C. elegans.

Metazoan cell death mechanisms are diverse and include numerous non-apoptotic programs. One program called entosis involves the invasion of live cells into their neighbors and is known to occur in cancers. Here, we identify a developmental function for entosis: to clear the male-specific linker cell in C. elegans. The linker cell leads migration to shape the gonad and is removed to facilitate fusion of the gonad to the cloaca. We find that the linker cell is cleared in a manner involving cell-cell adhesions and cell-autonomous control of uptake through linker cell actin. Linker cell entosis generates a lobe structure that is deposited at the site of gonad-to-cloaca fusion and is removed during mating. Inhibition of lobe scission inhibits linker cell death, demonstrating that the linker cell invades its host while alive. Our findings demonstrate a developmental function for entosis: to eliminate a migrating cell and facilitate gonad-to-cloaca fusion, which is required for fertility

The zebrafish as a novel model for the in vivo study of Toxoplasma gondii replication and interaction with 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 developed 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 brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo, we further analyzed Toxoplasma-macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo, suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites. Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity.This article has an associated First Person interview with the first author of the paper

Acute depletion of diacylglycerol from the Cis-Golgi affects localised nuclear envelope morphology during mitosis

Dysregulation of nuclear envelope (NE) assembly results in various cancers; for example, renal and some lung carcinomas ensue due to NE malformation. The NE is a dynamic membrane compartment and its completion during mitosis, is a highly regulated process but the detailed mechanism still remains incompletely understood. Previous studies have found isolated diacylglycerol (DAG) containing vesicles are essential for completing the fusion of NE in non-somatic cells. We investigated the impact of DAG depletion from cis-Golgi in mammalian cells on NE reassembly. Using advanced electron microscopy, we observed an enriched DAG population of vesicles at the vicinity of the NE gaps of telophase mammalian cells. We applied a miniSOG-C1-domain tag that localized DAG-enriched vesicles at the perinuclear region, which suggested the existence of NE fusogenic vesicles. We quantified the impact of Golgi-DAG depletion by measuring the in situ NE rim curvature of the re-forming NE. The rim curvature in these cells was significantly reduced compared with controls, which indicated a localized defect in NE morphology. Our novel results demonstrate the significance of the role of DAG from cis-Golgi for the regulation of NE assembly

Emergent mechanical control of vascular morphogenesis

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.

The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research