Ectocytosis renders T cell receptor signaling self-limiting at the immune synapse.

Cytotoxic T lymphocytes (CTLs) kill virus-infected and cancer cells through T cell receptor (TCR) recognition. How CTLs terminate signaling and disengage to allow serial killing has remained a mystery. TCR activation triggers membrane specialization within the immune synapse, including the production of diacylglycerol (DAG), a lipid that can induce negative membrane curvature. We found that activated TCRs were shed into DAG-enriched ectosomes at the immune synapse rather than internalized through endocytosis, suggesting that DAG may contribute to the outward budding required for ectocytosis. Budding ectosomes were endocytosed directly by target cells, thereby terminating TCR signaling and simultaneously disengaging the CTL from the target cell to allow serial killing. Thus, ectocytosis renders TCR signaling self-limiting.

Semantic segmentation of HeLa cells: An objective comparison between one traditional algorithm and four deep-learning architectures.

The quantitative study of cell morphology is of great importance as the structure and condition of cells and their structures can be related to conditions of health or disease. The first step towards that, is the accurate segmentation of cell structures. In this work, we compare five approaches, one traditional and four deep-learning, for the semantic segmentation of the nuclear envelope of cervical cancer cells commonly known as HeLa cells. Images of a HeLa cancer cell were semantically segmented with one traditional image-processing algorithm and four three deep learning architectures: VGG16, ResNet18, Inception-ResNet-v2, and U-Net. Three hundred slices, each 2000 × 2000 pixels, of a HeLa Cell were acquired with Serial Block Face Scanning Electron Microscopy. The first three deep learning architectures were pre-trained with ImageNet and then fine-tuned with transfer learning. The U-Net architecture was trained from scratch with 36, 000 training images and labels of size 128 × 128. The image-processing algorithm followed a pipeline of several traditional steps like edge detection, dilation and morphological operators. The algorithms were compared by measuring pixel-based segmentation accuracy and Jaccard index against a labelled ground truth. The results indicated a superior performance of the traditional algorithm (Accuracy = 99%, Jaccard = 93%) over the deep learning architectures: VGG16 (93%, 90%), ResNet18 (94%, 88%), Inception-ResNet-v2 (94%, 89%), and U-Net (92%, 56%).

M. tuberculosis infection of human iPSC-derived macrophages reveals complex membrane dynamics during xenophagy evasion.

Xenophagy is an important cellular defence mechanism against cytosol-invading pathogens, such as Mycobacterium tuberculosis (Mtb). Activation of xenophagy in macrophages targets Mtb to autophagosomes; however, how Mtb is targeted to autophagosomes in human macrophages at a high spatial and temporal resolution is unknown. Here, we use human induced pluripotent stem cell-derived macrophages (iPSDMs) to study the human macrophage response to Mtb infection and the role of the ESX-1 type VII secretion system. Using RNA-seq, we identify ESX-1-dependent transcriptional responses in iPSDMs after infection with Mtb. This analysis revealed differential inflammatory responses and dysregulated pathways such as eukaryotic initiation factor 2 (eIF2) signalling and protein ubiquitylation. Moreover, live-cell imaging revealed that Mtb infection in human macrophages induces dynamic ESX-1-dependent, LC3B-positive tubulovesicular autophagosomes (LC3-TVS). Through a correlative live-cell and focused ion beam scanning electron microscopy (FIB SEM) approach, we show that upon phagosomal rupture, Mtb induces the formation of LC3-TVS, from which the bacterium is able to escape to reside in the cytosol. Thus, iPSDMs represent a valuable model for studying spatiotemporal dynamics of human macrophage-Mtb interactions, and Mtb is able to evade capture by autophagic compartments.

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.

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.

Centriolar satellite- and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly.

Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction-induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring.

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.

Pelvic Nerve Injury Causes a Rapid Decrease in Expression of Choline Acetyltransferase and Upregulation of c-Jun and ATF-3 in a Distinct Population of Sacral Preganglionic Neurons.

Autonomic regulation of the urogenital organs is impaired by injuries sustained during pelvic surgery or compression of lumbosacral spinal nerves (e.g., cauda equina syndrome). To understand the impact of injury on both sympathetic and parasympathetic components of this nerve supply, we performed an experimental surgical and immunohistochemical study on adult male rats, where the structure of this complex part of the nervous system has been well defined. We performed unilateral transection of pelvic or hypogastric nerves and analyzed relevant regions of lumbar and sacral spinal cord, up to 4 weeks after injury. Expression of c-Jun, the neuronal injury marker activating transcription factor-3 (ATF-3), and choline acetyltransferase (ChAT) were examined. We found little evidence for chemical or structural changes in substantial numbers of functionally related but uninjured spinal neurons (e.g., in sacral preganglionic neurons after hypogastric nerve injury), failing to support the concept of compensatory events. The effects of injury were greatest in sacral cord, ipsilateral to pelvic nerve transection. Here, around half of all preganglionic neurons expressed c-Jun within 1 week of injury, and substantial ATF-3 expression also occurred, especially in neurons with complete loss of ChAT-immunoreactivity. There did not appear to be any death of retrogradely labeled neurons, in contrast to axotomy studies performed on other regions of spinal cord or sacral ventral root avulsion models. Each of the effects we observed occurred in only a subpopulation of preganglionic neurons at that spinal level, raising the possibility that distinct functional subgroups have different susceptibility to trauma-induced degeneration and potentially different regenerative abilities. Identification of the cellular basis of these differences may provide insights into organ-specific strategies for attenuating degeneration or promoting regeneration of these circuits after trauma.