Serial Block-face Scanning Electron Research Articles

Systematic Transmission Electron Microscopy‐Based Identification and 3D Reconstruction of Cellular Degradation Machinery (Adv. Biology 6/2023)

3D Reconstruction Macroautophagy is a dynamic process that regulates cellular mechanisms. It can be difficult to identify machinery and quantify them. In article 2200221, Antentor O. Hinton Jr. and co-workers demonstrate the use of transmission electron microscopy, immunogold labeling, immunofluorescence, and 3D reconstruction serial block face-scanning electron microscopy to identify and quantify lysosomes, autophagosomes, and lipid droplets under drug response conditions and experimental knockouts.

Serial Block Face-Scanning Electron Microscopy as a Burgeoning Technology.

Serial block face scanning electron microscopy (SBF-SEM), also referred to as serial block-face electron microscopy, is an advanced ultrastructural imaging technique that enables three-dimensional visualization that provides largerx- and y-axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF-SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004.Here, the authors provide an accessible overview of the advantages and challenges associated with SBF-SEM. Beyond this, the applications of SBF-SEM in biochemical domains as well as potential future clinical applications are briefly reviewed. Finally, the alternative forms of artificial intelligence-based segmentation which may contribute to devising a feasible workflow involving SBF-SEM, are also considered.

Exploring 3D leaf anatomical traits for C4 photosynthesis: chloroplast and plasmodesmata pit field size in maize and sugarcane.

Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C4 species, maize and sugarcane, with respect to cell surface area and cell volume. Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM. Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30-50% of mesophyll cell volume, and 60-70% of bundle sheath cell volume. Roughly 2-3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells. This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C4 photosynthesis.

Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector.

Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion,which are critical for a holistic understanding of the Leishmania life cycle.

Prolonged exposure of neonatal mice to sevoflurane leads to hyper-ramification in microglia, reduced contacts between microglia and synapses, and defects in adult behavior.

Prolonged exposure to general anesthetics during development is known to cause neurobehavioral abnormalities, but the cellular and molecular mechanisms involved are unclear. Microglia are the resident immune cells in the central nervous system and play essential roles in normal brain development. In the study, postnatal day 7 (P7) C57BL/6 mice were randomly assigned to two groups. In the sevoflurane (SEVO), mice were exposed to 2.5% sevoflurane for 4 h. In the control group, mice were exposed to carrier gas (30% O2/70% N2) for 4 h. Fixed brain slices from P14 to P21 mice were immunolabeled for ionized calcium-binding adapter molecule 1 (IBA-1) to visualize microglia. The morphological analysis of microglia in the somatosensory cortex was performed using ImageJ and Imaris software. Serial block face scanning electron microscopy (SBF-SEM) was performed to assess the ultrastructure of the microglia and the contacts between microglia and synapse in P14 and P21 mice. The confocal imaging of brain slices was performed to assess microglia surveillance in resting and activated states in P14 and P21 mice. Behavioral tests were used to assess the effect of microglia depletion and repopulation on neurobehavioral abnormalities caused by sevoflurane exposure. The prolonged exposure of neonatal mice to sevoflurane induced microglia hyper-ramification with an increase in total branch length, arborization area, and branch complexity 14 days after exposure. Prolonged neonatal sevoflurane exposure reduced contacts between microglia and synapses, without affecting the surveillance of microglia in the resting state or responding to laser-induced focal brain injury. These neonatal changes in microglia were associated with anxiety-like behaviors in adult mice. Furthermore, microglial depletion before sevoflurane exposure and subsequent repopulation in the neonatal brain mitigated anxiety-like behaviors caused by sevoflurane exposure. Our experiments indicate that general anesthetics may harm the developing brain, and microglia may be an essential target of general anesthetic-related developmental neurotoxicity.

Refractile bodies of Eimeria tenella are proteinaceous membrane-less organelles that undergo dynamic changes during infection.

Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of Eimeria, parasitic coccidian protozoa. High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells. Live cell imaging of MDBK cells infected with E. tenella sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs via merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape. MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.

Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery.

Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer-scale micrographs of cellular degradation components in a fixed sample. Serial block face-scanning electron microscopy is also used to visualize the3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1.

3D reconstruction shows mouse skeletal muscle may decline in function across aging due to the MICOS complex

Mitochondria are active organelles, undergoing harmonized cycles of fission and fusion, driven by ‘mitochondrial dynamics,’ to retain their morphology, distribution, and size. Mitochondrial dynamics has emerged as a critical process in maintaining cellular homeostasis. Interestingly, it has been long respected that a decrease in mitochondrial function escorts aging in specific tissues. For example, skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. This decrease might, in turn, contribute to the observed age-dependent decline in organ function by mutations or alterations in mitochondrial fusion and fission proteins associated with several diseases. However, new players in regulating mitochondrial shape and cristae shape have been recently studied, such as the mitochondrial contact site and cristae organizing system (MICOS) complex. The MICOS complex is a multi-protein interaction hub that helps define cristate and mitochondria architecture. However, the MICOS complex has not been implicated in regulating organelle structure changes during aging. Thus, we hypothesized that loss of the MICOS complex during aging may increase mitochondrial fragmentation, decrease nanotunnels, and alter cristae morphology. To do this, we examined the three-dimensional morphology of mitochondria networks in young (3-month) and aged (2-year) murine gastrocnemius muscle via serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. We found differences in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, MICOS dynamics, and gene expression in skeletal muscle during aging. We also found an association between OPA-1 and the MICOS complex in the gastrocnemius with mitochondrial aging. Furthermore, the loss of the MICOS complex was linked with decreased oxidative capacity and altered mitochondrial metabolism. Potentially, this suggests a novel relationship between the MICOS complex and aging in skeletal muscle.

Myelination generates aberrant ultrastructure that is resolved by microglia.

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one "eat me" signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement.

Correlative multiscale microCT-SBF-SEM imaging of resin-embedded tissue.

Three-dimensional biological microscopy presents a trade-off between spatial resolution and field of view. Correlative approaches applying multiple imaging techniques to the same sample can therefore mitigate against these trade-offs. Here, we present a workflow for correlative microscopic X-ray microfocus computed tomography (microCT) and serial block face scanning electron microscopy (SBF-SEM) imaging of resin-embedded tissue, using mammalian placental tissue samples as an example. This correlative X-ray and electron microscopy (CXEM) workflow allows users to image the same sample at multiple resolutions, and target the region of interest (ROI) for SBF-SEM based on microCT. We detail the protocols associated with this workflow and demonstrate its application in multiscale imaging of horse placental villi and ROI selection in the labyrinthine zone of a mouse placenta. These examples demonstrate how the protocol may need to be adapted for tissues with different densities.

Automated large volume sample preparation for vEM.

New developments in electron microscopy technology, improved efficiency of detectors, and artificial intelligence applications for data analysis over the past decade have increased the use of volume electron microscopy (vEM) in the life sciences field. Moreover, sample preparation methods are continuously being modified by investigators to improve final sample quality, increase electron density, combine imaging technologies, and minimize the introduction of artifacts into specimens under study. There are a variety of conventional bench protocols that a researcher can utilize, though most of these protocols require several days. In this work, we describe the utilization of an automated specimen processor, the mPrep™ ASP-2000™, to prepare samples for vEM that are compatible with focused ion beam scanning electron microscopy (FIB-SEM), serial block face scanning electron microscopy (SBF-SEM), and array tomography (AT). The protocols described here aimed for methods that are completed in a much shorter period of time while minimizing the exposure of the operator to hazardous and toxic chemicals and improving the reproducibility of the specimens' heavy metal staining, all without compromising the quality of the data acquired using backscattered electrons during SEM imaging. As a control, we have included a widely used sample bench protocol and have utilized it as a comparator for image quality analysis, both qualitatively and using image quality analysis metrics.

Ultrastructural effects of sleep and wake on the parallel fiber synapses of the cerebellum.

Multiple evidence in rodents shows that the strength of excitatory synapses in the cerebral cortex and hippocampus is greater after wake than after sleep. The widespread synaptic weakening afforded by sleep is believed to keep the cost of synaptic activity under control, promote memory consolidation, and prevent synaptic saturation, thus preserving the brain's ability to learn day after day. The cerebellum is highly plastic and the Purkinje cells, the sole output neurons of the cerebellar cortex, are endowed with a staggering number of excitatory parallel fiber synapses. However, whether these synapses are affected by sleep and wake is unknown. Here, we used serial block face scanning electron microscopy to obtain the full 3D reconstruction of more than 7000 spines and their parallel fiber synapses in the mouse posterior vermis. This analysis was done in mice whose cortical and hippocampal synapses were previously measured, revealing that average synaptic size was lower after sleep compared to wake with no major changes in synapse number. Here, instead, we find that while the average size of parallel fiber synapses does not change, the number of branched synapses is reduced in half after sleep compared to after wake, corresponding to ~16% of all spines after wake and ~8% after sleep. Branched synapses are harbored by two or more spines sharing the same neck and, as also shown here, are almost always contacted by different parallel fibers. These findings suggest that during wake, coincidences of firing over parallel fibers may translate into the formation of synapses converging on the same branched spine, which may be especially effective in driving Purkinje cells to fire. By contrast, sleep may promote the off-line pruning of branched synapses that were formed due to spurious coincidences.

A Putative Erythroid Synapse Linking Developing Erythroblasts and Macrophages in Erythroblastic Islands

An estimated 25% of the global population suffer from anemia, a major cause being deficient erythropoiesis. Mammalian erythropoiesis in the adult occurs primarily in the bone marrow (BM), with the spleen being an active site of extramedullary erythropoiesis during anemic stress. Here, we examine regulators of the anemic stress response using an unbiased transcriptomic approach to compare FACS-sorted splenic Ter-119+ erythroid precursors, from untreated mice or phenylhydrazine (PHZ)-treated mice exhibiting chemically-induced anemia. Transcriptomic analyses revealed unexpected expression of a broad range of immune markers in untreated mouse splenic erythroid cells, most which were conspicuously diminished in expression during PHZ-induced anemic stress. These surface antigens, previously unreported on erythroid cells, include markers of B lymphocytes (CD22, CD74), T lymphocytes (CD2, CD3ε) and leukocytes including CD11a/LFA-1, CD45, CD48, CD53, as well as the homing receptors L-selectin and PSGL-1. Immunophenotyping confirmed expression of these immune markers on a subpopulation of steady state BM and splenic erythroid precursors, with a significant reduction in immune marker-expressing erythroid cells seen in the spleen during anemic stress. Erythropoiesis is governed by a close relationship between erythroid precursors and central macrophages in multicellular clusters termed erythroblastic islands (EBIs). Confocal microscopy demonstrated localization of the immune markers CD2, CD3ε, CD11a and PSGL-1 on Ter-119+ mouse erythroid precursors with frequent clustering of markers seen at intercellular contact points where erythroid cells interact with the central EBI macrophage. PHZ-treated mice exhibited a significantly reduced frequency of EBIs in both the BM and spleen compared to control mice. PHZ-treated mice also demonstrated downregulated Interferon Regulatory Factor-8 (Irf8) transcripts on transcriptomic analysis of splenic erythroid precursors. Irf8-deficient mice have been reported to have a profoundly reduced erythroid compartment. There are also reports that Irf8 may be a regulator of macrophage morphology. We therefore studied Irf8-deficient mice to evaluate whether Irf8 has a role in EBI-regulation. BM analysis of Irf8-deficient mice showed a reduced frequency of EBI-like structures compared to wildtype (WT) mice. Scanning electron micrographs revealed that Irf8-/- macrophages are smaller with fewer erythroid precursors attached compared to WT macrophages. Irf8-/- EBIs harbored no identifiable reticulocytes in contrast to WT EBIs. EBI rosette reconstitution assays demonstrated that Irf8-/- erythroid precursors were unable to bind to WT or Irf8-/- macrophages, whereas Irf8-/- macrophages could interact with WT erythroid cells. Immunophenotyping of Irf8-/- erythroid precursors revealed that immune marker expression (CD2, CD38, CD43, CD45, PSGL-1) was significantly lower compared to WT counterparts. These data suggest that Irf8 may regulate both macrophage morphology and erythroid cell surface phenotype influencing erythropoiesis. The newly uncovered immune markers expressed by erythroid cells are also involved in formation of immunological synapses between lymphocytes and macrophages. This led us to question whether an analogous synapse-like structure may exist in EBIs. Serial block face scanning electron microscopy (SBF-SEM) was used to assess EBI morphology at the nanometer scale. SBF-SEM micrographs show erythroblasts extending pseudopods towards the macrophage for attachment. Three-dimensional volumetric reconstruction of SBF-SEM micrographs revealed en face spatial topologies present on the surface of EBI central macrophages similar to those seen in immunological synapses. Our findings suggest that immune markers, possibly mediated by Irf8, may function to form synaptic-like interactions between erythroid cells and macrophages that result in EBIs - an "erythroid synapse". Failure in forming these interactions may result in alterations in EBI formation with potential downstream impacts on erythropoiesis. Improved understanding of how EBIs form and function may aid in the development of therapeutic agents to combat anemia.

A fibre tracking algorithm for volumetric microstructural data - application to tendons

Tendons are crucial connective tissues made almost entirely of bundles of long, near-parallel collagen fibrils, and are vitally important to skeletal stability and mechanical function. Tendon structure is typically quantified in 2D, whereas, in this work, we have used serial block face-scanning electron microscopy to image tendons in 3D. We present a custom fibre tracking algorithm (FTA), with which we have characterised the 3D microstructure of tendon. Currently available tools for fibre tracing were unsuitable for tracking large numbers of fibrils and handling imaging artefacts associated with EM. We have tracked fibrils through a representative tendon volume and measured their relative length, diameter, orientation, chirality, tortuosity and volume fraction, which are just some of the measurements it is possible to make with the FTA depending on the research question. This algorithm has been developed in a general way and can be applied to a range of biological research questions relating to tendon structure-function relationships, on topics such as ageing, disease, development and injury. The FTA is also applicable to other fibrous biological materials, as well as engineered materials and textiles; it is written using Python and is freely available to download. Statement of significanceWe have created an algorithm for tracking fibres in 3D image stacks and applied it to tendon tissue. Previous studies have examined tendon structure in 2D, whereas we have imaged tendons in 3D using volumetric electron microscopy. Currently available fibre tracing tools could not track the large numbers of fibres or tolerate the artefacts present in biological imaging data. Using our algorithm, we have reconstructed and characterised the geometrical properties of the collagen fibrils (length, width, alignment, area, location). This algorithm could help to answer questions in biology which relate tissue microstructure to function in areas such as ageing, disease, development and injury. It could also be used to study engineered materials and textiles and is available freely to download.

Serial block face scanning electron microscopy reveals region-dependent remodelling of transverse tubules post-myocardial infarction.

The highly organized transverse tubule (t-tubule) network facilitates cardiac excitation–contraction coupling and synchronous cardiac myocyte contraction. In cardiac failure secondary to myocardial infarction (MI), changes in the structure and organization of t-tubules result in impaired cardiac contractility. However, there is still little knowledge on the regional variation of t-tubule remodelling in cardiac failure post-MI. Here, we investigate post-MI t-tubule remodelling in infarct border and remote regions, using serial block face scanning electron microscopy (SBF-SEM) applied to a translationally relevant sheep ischaemia reperfusion MI model and matched controls. We performed minimally invasive coronary angioplasty of the left anterior descending artery, followed by reperfusion after 90 min to establish the MI model. Left ventricular tissues obtained from control and MI hearts eight weeks post-MI were imaged using SBF-SEM. Image analysis generated three-dimensional reconstructions of the t-tubular network in control, MI border and remote regions. Quantitative analysis revealed that the MI border region was characterized by t-tubule depletion and fragmentation, dilation of surviving t-tubules and t-tubule elongation. This study highlights region-dependent remodelling of the tubular network post-MI and may provide novel localized therapeutic targets aimed at preservation or restoration of the t-tubules to manage cardiac contractility post-MI.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

P192 Investigating the link between pleomorphism and virulence in Cryptococcus

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesFungal pathogens Cryptococcus neoformans and C. gattii are responsible for hundreds of thousands of annual deaths in immunocompromised individuals. Considerable phenotypic variation is exhibited by strains in response to stresses encountered during host infection, including increased capsule and cell size, the release of shed capsule, and the production of giant (>15 μm), micro (<1 μm), and irregular cells. We aimed to investigate whether the production of these morphological variants is associated with virulence using two sets of strains. The first is a collection of diverse clinical isolates obtained from HIV/AIDS patients in Botswana with accompanying clinical data. The second is a collection of lineages derived from the C. neoformans type strain H99 with high genetic similarity but differing levels of virulence. Some lineages in this set possess a mutation in SGF29, which encodes a component of the SAGA histone acetylation complex that has previously been implicated in their hypervirulence.MethodsIsolates were cultured under conditions that simulate stresses encountered in vivo (DMEM, 5% CO2, 37°C) as these are known to enhance capsule production and induce cell size changes. Cells were counterstained with India Ink, visualized by light microscopy, and phenotypes were scored. For clinical isolates, MLST analysis was performed to determine their relatedness. For H99 strains, Galleria mellonella larval infection assays, growth curves, and antifungal susceptibility testing was performed to confirm their relative virulence and growth profiles. Serial block face and regular scanning electron microscopy were used to investigate the internal morphology of the giant, micro, and irregular cells to confirm that they possess attributes of functional cells.ResultsSubstantial pleomorphism was seen across both collections. In the clinical strain set, phenotypic variables fell into two groups associated with differing symptoms. The production of ‘large’ phenotypes was associated with a higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting that these are induced in early-stage infection. ‘Small’ phenotypes were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators, and positively correlated with intracranial pressure indicators, suggesting that they are produced later during infection and may promote proliferation and dissemination. Isolates possessing giant cells, microcells, and shed capsule were rare, but strikingly, they were associated with patient death.In the H99 set, strains from hypervirulent lineages had larger average capsule size, greater variation in cell size, and increased production of microcells and shed capsule. Deletion of SGF29 in an intermediate virulence lineage substantially increased its production of microcells and released capsule, consistent with a switch to hypervirulence. SGF29 loss-of-function mutations were subsequently identified in clinical isolates and were found to be significantly correlated with patient death. Expansion of a TA repeat in the second intron of SGF29 in clinical isolates was positively correlated with cell and capsule size, suggesting it also affects SGF29 function.ConclusionOur results extend the evidence for a link between pleomorphism and virulence, with a likely role for epigenetic mechanisms mediated by SAGA-induced histone acetylation.

S5.3c Investigating the link between pleomorphism and virulence in Cryptococcus

S5.3 Cellular pleomorphism and fungal virulence, September 22, 2022, 3:00 PM - 4:30 PMObjectivesFungal pathogens Cryptococcus neoformans and Cryptococcus gattii are responsible for hundreds of thousands of annual deaths in immunocompromised individuals. Considerable phenotypic variation is exhibited by strains in response to stresses encountered during host infection, including increased capsule and cell size, the release of shed capsule, and the production of giant (> 15 μm), micro (< 1 μm), and irregular cells. We aimed to investigate whether the production of these morphological variants is associated with virulence using two sets of strains. The first is a collection of diverse clinical isolates obtained from HIV/AIDS patients in Botswana with accompanying clinical data. The second is a collection of lineages derived from the C. neoformans type strain H99 with high genetic similarity but differing levels of virulence. Some lineages in this set possess a mutation in SGF29, which encodes a component of the SAGA histone acetylation complex that has previously been implicated in their hypervirulence.MethodsIsolates were cultured under conditions that simulate stresses encountered in vivo (DMEM, 5% CO2, 37°C) as these are known to enhance capsule production and induce cell size changes. Cells were counterstained with india ink, visualized by light microscopy, and phenotypes were scored. For clinical isolates, MLST analysis was performed to determine their relatedness. For H99 strains, Galleria mellonella larval infection assays, growth curves, and antifungal susceptibility testing was performed to confirm their relative virulence and growth profiles. Serial block face and regular scanning electron microscopy were used to investigate the internal morphology of the giant, micro, and irregular cells to confirm that they possess attributes of functional cells.ResultsSubstantial pleomorphism was seen across both collections. In the clinical strain set, phenotypic variables fell into two groups associated with differing symptoms. The production of ‘large’ phenotypes was associated with a higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting that these are induced in early-stage infection. ‘Small’ phenotypes were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators, and positively correlated with intracranial pressure indicators, suggesting that they are produced later during infection and may promote proliferation and dissemination. Isolates possessing giant cells, microcells, and shed capsules were rare, but strikingly, they were associated with patient death.In the H99 set, strains from hypervirulent lineages had larger average capsule size, greater variation in cell size, and increased production of microcells and shed capsules. Deletion of SGF29 in an intermediate virulence lineage substantially increased its production of microcells and released capsule, consistent with a switch to hypervirulence. SGF29 loss-of-function mutations were subsequently identified in clinical isolates and were found to be significantly correlated with patient death. Expansion of a TA repeat in the second intron of SGF29 in clinical isolates was positively correlated with cell and capsule size, suggesting it also affects Sgf29 function.ConclusionOur results extend the evidence for a link between pleomorphism and virulence, with a likely role for epigenetic mechanisms mediated by SAGA-induced histone acetylation.

Budding pouches and associated bubbles: 3D visualization of exo-membrane structures in plasmodium falciparum gametocytes.

Plasmodium falciparum gametocytes have unique morphology, metabolism, and protein expression profiles in their asexual stages of development. In addition to the striking changes in their appearance, a wide variety of “exo-membrane structures” are newly formed in the gametocyte stage. Little is known about their function, localization, or three-dimensional structural information, and only some structural data, typically two-dimensional, have been reported using conventional electron microscopy or fluorescence microscopy. For better visualization of intracellular organelle and exo-membrane structures, we previously established an unroofing technique to directly observe Maurer’s clefts (MCs) in asexual parasitized erythrocytes by removing the top part of the cell’s membrane followed by transmission electron microscopy. We found that MCs have numerous tethers connecting themselves to the host erythrocyte membrane skeletons. In this study, we investigated the intracellular structures of gametocytes using unroofing-TEM, Serial Block Face scanning electron microscopy, and fluorescence microscopy to unveil the exo-membrane structures in gametocytes. Our data showed “balloon/pouch”-like objects budding from the parasitophorous vacuole membrane (PVM) in gametocytes, and some balloons included multiple layers of other balloons. Furthermore, numerous bubbles appeared on the inner surface of the erythrocyte membrane or PVM; these were similar to MC-like membranes but were smaller than asexual MCs. Our study demonstrated P. falciparum reforms exo-membranes in erythrocytes to meet stage-specific biological activities during their sexual development.

Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion.

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there are few data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised apical secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites; we report a detailed dissection of the three- dimensional organisation of the conoid and show there is high curvature of the tubulin-containing fibres that might be linked to the unusual comma-shaped arrangement of protofilaments. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and microtubule-associated vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins may be trafficked between the parasite and the host cytoplasm.

An ultrastructural study of the deep pineal gland of the Sprague Dawley rat using transmission and serial block face scanning electron microscopy: cell types, barriers, and innervation.

The morphology of the deep pineal gland of the Sprague Dawley rat was investigated by serial block face scanning electron microscopy. Cells were three-dimensionally (3-D) reconstructed using the software Fiji TrackEM. The deep pineal gland consisted of 2-5 layers of electron-lucent pinealocytes, with a euchromatic nucleus, endowed with one or two processes. Laterally, the deep pineal merged with the habenula and the stria medullaris thalami, via an intermediate area containing cells with more electron-dense cytoplasm and an indented nucleus with heterochromatin. Neither nerve terminals nor capillaries were observed in the deep pineal itself but present in the intermediate parts of the gland. The deep pineal was in contact with the third ventricle via the pineal and suprahabenular recesses. The ependymal lining in these recesses was an epithelium connected by tight junctions between their lateral cell membranes. Several intraventricular nerve terminals were in contact with the ependyma. 3-D reconstructions showed the ependymal cells endowed with long slender process penetrating the underlying pineal parenchyma. Few "tanocyte-like" ependymal cells, endowed with a process, reaching the subarachnoid space on the inferior surface of the deep pineal were observed. In addition, pinealocyte and astrocyte processes, often connected by gap junctions, bordered the inferior surface. In summary, the rat deep pineal gland is a neuroendocrine structure connected to the habenula. We here report specialized ependymal cells that might transmit signals from the cerebrospinal fluid to the deep pineal parenchyma and a "trans-pineal tanocyte-like cell" that connects the ventricular system with the subarachnoid space.