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A transmission electron microscope (TEM) enables the magnification and visualisation of cell and tissue ultrastructure that cannot otherwise be resolved using a light microscope. In transmission electron microscopy, a high energy electron beam generated from an electron gun is focused by electromagnetic lenses onto an ultrathin section of plastic embedded specimen. Electrons are transmitted through the specimen onto a fluorescent screen based on sample density, and an image is acquired by a digital camera. Here, a protocol for preparing tissue specimens for TEM ultrastructural analysis is described. A tissue specimen is fixed, dehydrated and infiltrated with resin. The resin is then polymerised, and embedded tissue sectioned using an ultramicrotome. Ultrathin sections are contrasted with heavy metals prior to imaging using a TEM. Options for faster processing using a Microwave processor are described for relevant steps.

Ultrastructural morphological information is routinely obtained from the processing of cells and tissue into plastics for transmission electron microscopy imaging. This technique involves the use of strong chemical fixatives, solvents and resins, which destroy the antigenicity of samples. Freezing samples to a vitreous state using cryo-electron microscopy techniques preserves ultrastructure without the use of harsh chemicals, however, immunolabelling cannot be achieved at temperatures as low as -196 degrees Celsius. Here we describe a protocol for the Tokuyasu technique for cryopreservation and immunolabelling of subcellular structures in cells. Cells are lightly chemically fixed and cryoprotected in sucrose before freezing in liquid nitrogen. Cells are sectioned by cryo-ultramicrotomy and retrieved cryo-sections are thawed. Thawed cryo-sections are then immunolabelled, stained and imaged using a transmission electron microscope.

A transmission electron microscope (TEM) enables the magnification and visualisation of cell and tissue ultrastructure that cannot otherwise be resolved using a light microscope. In transmission electron microscopy, a high energy electron beam generated from an electron gun is focused by electromagnetic lenses onto an ultrathin section of plastic embedded specimen. Electrons are transmitted through the specimen onto a fluorescent screen based on sample density, and an image acquired by a digital camera. Here, a protocol for preparing cells for TEM ultrastructural analysis is described. A cell sample is fixed, dehydrated and infiltrated with resin. The resin is then polymerised, and embedded cell sample sectioned using an ultramicrotome. Ultrathin sections are contrasted with heavy metals prior to imaging using a TEM. Options for faster processing using a Microwave processor are described for relevant steps.

Immuno- correlative light and electron microscopy (iCLEM) combines ultrastructural information obtained from high resolution electron microscopy and the use of genetically encoded or cytochemical markers. Immuno-CLEM takes advantage of the antigenicity preserved by Tokuyasu sample preparation to identify, quantify and characterise heterogeneous cell populations in small organisms, organs and tissue of healthy and diseased states. iCLEM can be used in combination with scanning EM (SEM), scanning TEM (STEM), and transmission EM (TEM). These protocols are well-suited, for example, for investigating neural stem and progenitor cell populations of the vertebrate nerve system and are available as separate protocols on protocol.io. Here, a method for iCLEM-STEM is described using an adult zebrafish telencephalon brain as a model. This organ is small in size allowing the complete dorsal telencephalic niche to be visualised in sections, and has diverse cell profiles and regenerative potential of local neural stem and progenitor cells. iCLEM-STEM involves the examination of ultrathin tissue sections (62-70 nm) using immunofluorescence labelling and subsequent SEM imaging to obtain a high resolution overview of these sections with greater morphological detail compared to iCLEM-SEM. This protocol should be of particular interest to EM facilities with SEM, but not TEM access.

The dendritic cell receptor Clec9A facilitates processing of dead cell-derived antigens for cross-presentation and the induction of effective CD8+ T cell immune responses. Here, we show that this process is regulated by E3 ubiquitin ligase RNF41 and define a new ubiquitin-mediated mechanism for regulation of Clec9A, reflecting the unique properties of Clec9A as a receptor specialized for delivery of antigens for cross-presentation. We reveal RNF41 is a negative regulator of Clec9A and the cross-presentation of dead cell-derived antigens by mouse dendritic cells. Intriguingly, RNF41 regulates the downstream fate of Clec9A by directly binding and ubiquitinating the extracellular domains of Clec9A. At steady-state, RNF41 ubiquitination of Clec9A facilitates interactions with ER-associated proteins and degradation machinery to control Clec9A levels. However, Clec9A interactions are altered following dead cell uptake to favor antigen presentation. These findings provide important insights into antigen cross-presentation and have implications for development of approaches to modulate immune responses.

Protein targeting into mitochondria from the cytoplasm is fundamental to the cell biology of all eukaryotes. Our understanding of this process is heavily biased towards "model" organisms, such as animals and fungi, and it is less clear how conserved this process is throughout diverse eukaryotes. In this study, we have surveyed mitochondrial protein sorting signals from a representative of the dinoflagellate algae. Dinoflagellates are a phylum belonging to the group Alveolata, which also includes apicomplexan parasites and ciliates. We generated 46 mitochondrial gene sequences from the dinoflagellate Karlodinium micrum and analysed these for mitochondrial sorting signals. Most of the sequences contain predicted N-terminal peptide extensions that conform to mitochondrial targeting peptides from animals and fungi in terms of length, amino acid composition, and propensity to form amphipathic α-helices. The remainder lack predicted mitochondrial targeting peptides and represent carrier proteins of the inner mitochondrial membrane that have internal targeting signals in model eukaryotes. We tested for functional conservation of the dinoflagellate mitochondrial sorting signals by expressing K. micrum mitochondrial proteins in the fungus Saccharomyces cerevisiae. Both the N-terminal and internal targeting signals were sufficiently conserved to operate in this distantly related system. This study indicates that the character of mitochondrial sorting signals was well established prior to the radiation of major eukaryotic lineages and has shown remarkable conservation during long periods of evolution.

The effective engagement of cytotoxic lymphocytes (CLs) with their target cells is essential for the removal of virus-infected and malignant cells from the body. The spatiotemporal properties that define CL engagement and killing of target cells remain largely uncharacterized due to a lack of biological reporters. We have used a novel live cell microscopy technique to visualize the engagement of primary human and mouse CL with their targets and the subsequent delivery of the lethal hit. Extensive quantitative real-time analysis of individual effector–target cell conjugates demonstrated that a single effector calcium flux event was sufficient for the degranulation of human CLs, resulting in the breach of the target cell membrane by perforin within 65–100 s. In contrast, mouse CLs demonstrated distinct calcium signaling profiles leading to degranulation: whereas mouse NKs required a single calcium flux event, CD8+ T cells typically required several calcium flux events before perforin delivery. Irrespective of their signaling profile, every target cell that was damaged by perforin died by apoptosis. To our knowledge, we demonstrate for the first time that perforin pore delivery is unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin resistant, as intact CTLs presented as targets to effector CTLs were capable of being killed by perforin-dependent mechanisms. Our results highlight the remarkable efficiency and specificity of perforin pore delivery by CLs.

Mitochondrial metabolism is central to the supply of ATP and numerous essential metabolites in most eukaryotic cells. Across eukaryotic diversity, however, there is evidence of much adaptation of the function of this organelle according to specific metabolic requirements and/or demands imposed by different environmental niches. This includes substantial loss or retailoring of mitochondrial function in many parasitic groups that occupy potentially nutrient-rich environments in their metazoan hosts. Infrakingdom Alveolata comprises a well-supported alliance of three disparate eukaryotic phyla-dinoflagellates, apicomplexans, and ciliates. These major taxa represent diverse lifestyles of free-living phototrophs, parasites, and predators and offer fertile territory for exploring character evolution in mitochondria. The mitochondria of apicomplexan parasites provide much evidence of loss or change of function from analysis of mitochondrial protein genes. Much less, however, is known of mitochondrial function in their closest relatives, the dinoflagellate algae. In this study, we have developed new models of mitochondrial metabolism in dinoflagellates based on gene predictions and stable isotope labeling experiments. These data show that many changes in mitochondrial gene content previously only known from apicomplexans are found in dinoflagellates also. For example, loss of the pyruvate dehydrogenase complex and changes in tricarboxylic acid (TCA) cycle enzyme complement are shared by both groups and, therefore, represent ancestral character states. Significantly, we show that these changes do not result in loss of typical TCA cycle activity fueled by pyruvate. Thus, dinoflagellate data show that many changes in alveolate mitochondrial metabolism are independent of the major lifestyle changes seen in these lineages and provide a revised view of mitochondria character evolution during evolution of parasitism in apicomplexans.

Most photosynthetic dinoflagellates harbour a red alga-derived secondary plastid. In the dinoflagellate Karlodinium micrum, this plastid was replaced by a subsequent endosymbiosis, resulting in a tertiary plastid derived from a haptophyte. Evolution of endosymbionts entails substantial relocation of endosymbiont genes to the host nucleus: a process called endosymbiotic gene transfer (EGT). In K. micrum, numerous plastid genes from the haptophyte nucleus are found in the host nucleus, providing evidence for EGT in this system. In other cases of endosymbiosis, notably ancient primary endosymbiotic events, EGT has been inferred to contribute to remodeling of other cell functions by expression of proteins in compartments other than the endosymbiont from which they derived. K. micrum provides a more recently derived endosymbiotic system to test for evidence of EGT and gain of function in non-plastid compartments. In this study, we test for gain of haptophyte-derived proteins for mitochondrial function in K. micrum. Using molecular phylogenies we have analysed whether nucleus-encoded mitochondrial proteins were inherited by EGT from the haptophyte endosymbiont, or vertically inherited from the dinoflagellate host lineage. From this dataset we found no evidence of haptophyte-derived mitochondrial genes, and the only cases of non-vertical inheritance were genes derived from lateral gene transfer events.

Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm) of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3′-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation) domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.