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Nuclear morphology of carcinoma cells is critical for the pathological diagnosis of papillary thyroid carcinoma (PTC). However, three-dimensional architecture of PTC nuclei is still elusive. In this study, we analyzed the three-dimensional ultrastructure of PTC nuclei using serial block-face scanning electron microscopy which takes advantage of the high-throughput acquisition of serial electron microscopic images and three-dimensional reconstruction of subcellular structures. En bloc-stained and resin-embedded specimens were prepared from surgically removed PTCs and normal thyroid tissues. We acquired two-dimensional images from serial block-face scanning electron microscopy and reconstructed three-dimensional nuclear structures. Quantitative comparisons showed that the nuclei of carcinoma cells were larger and more complex than those of normal follicular cells. The three-dimensional reconstruction of carcinoma nuclei divided intranuclear cytoplasmic inclusions into "open intranuclear cytoplasmic inclusions" connecting to cytoplasm outside the nucleus and "closed intranuclear cytoplasmic inclusions" without that connection. Cytoplasm with abundant organelles was observed in open inclusions, but closed inclusions contained fewer organelles with or without degeneration. Granules with a dense core were only observed in closed inclusions. Our observations suggested that open inclusions originate from nuclear invaginations, and disconnection from cytoplasm leads to closed inclusions., (© 2023 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Eukaryotic cells compartmentalize their internal milieu in order to achieve specific reactions in time and space. This organization in distinct compartments is essential to allow subcellular processing of regulatory signals and generate specific cellular responses. In the nucleus, genetic information is packaged in the form of chromatin, an organized and repeated nucleoprotein structure that is a source of epigenetic information. In addition, cells organize the distribution of macromolecules via various membrane-less nuclear organelles, which have gathered considerable attention in the last few years. The macromolecular multiprotein complexes known as Promyelocytic Leukemia Nuclear Bodies (PML NBs) are an archetype for nuclear membrane-less organelles. Chromatin interactions with nuclear bodies are important to regulate genome function. In this review, we will focus on the dynamic interplay between PML NBs and chromatin. We report how the structure and formation of PML NBs, which may involve phase separation mechanisms, might impact their functions in the regulation of chromatin dynamics. In particular, we will discuss how PML NBs participate in the chromatinization of viral genomes, as well as in the control of specific cellular chromatin assembly pathways which govern physiological mechanisms such as senescence or telomere maintenance., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Despite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected role of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals distinct functions of the two RRMs in TDP-43 NB formation. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we discover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules, which become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Essential tremor is one of the most common movement disorders. Despite its high prevalence and heritability, the genetic aetiology of essential tremor remains elusive. Up to now, only a few genes/loci have been identified, but these genes have not been replicated in other essential tremor families or cohorts. Here we report a genetic study in a cohort of 197 Chinese pedigrees clinically diagnosed with essential tremor. Using a comprehensive strategy combining linkage analysis, whole-exome sequencing, long-read whole-genome sequencing, repeat-primed polymerase chain reaction and GC-rich polymerase chain reaction, we identified an abnormal GGC repeat expansion in the 5' region of the NOTCH2NLC gene that co-segregated with disease in 11 essential tremor families (5.58%) from our cohort. Clinically, probands that had an abnormal GGC repeat expansion were found to have more severe tremor phenotypes, lower activities of daily living ability. Obvious genetic anticipation was also detected in these 11 essential tremor-positive families. These results indicate that abnormal GGC repeat expansion in the 5' region of NOTCH2NLC gene is associated with essential tremor, and provide strong evidence that essential tremor is a family of diseases with high clinical and genetic heterogeneities., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Five chimney swift fledglings died following a progressive loss of appetite and condition while being cared for by an experienced wildlife rehabilitator. All animals had severe necrotizing and heterophilic ventriculitis, with myriad epithelial cells characterized by karyomegaly with intranuclear inclusion bodies. Transmission electron microscopy showed distention of epithelial cell nuclei and chromatin peripheralization by nonenveloped, icosahedral, 75- to 85-nm-diameter virions. Degenerate nested PCR for a highly conserved region of the adenovirus DNA polymerase gene was positive. BLAST analysis of the amplicon sequence indicated the presence of a novel adenovirus, with 74% homology to Antarctic penguin adenoviruses and 72% homology to a bat adenovirus, at low query coverages of only 65% and 63%, respectively. BLAST analysis of the predicted amino acid sequence generated the highest scores for squamate adenoviruses at 100% query coverage. Based on phylogenetic analysis of the partial amino acid sequence of the DNA polymerase, the chimney swift virus was a novel adenovirus most closely related to the Atadenovirus genus. Using a probe based on the novel viral sequence, DNA in situ hybridization identified viral nucleic acid in the nucleus. While the tentatively named chimney swift adenovirus-1 (CsAdV-1) is so far classified with the Atadenoviruses, it is relatively divergent from other members of that genus and may represent the first identified member of a new genus of Adenoviruses.

For decades, intranuclear inclusions in many normal and neoplastic cells have been considered to be mere invaginations of cytoplasm into the nucleus without any notable function or influence on disease. We investigated such inclusions in 75 specimens of hepatocellular carcinoma (HCC). In this context we demonstrate that these inclusions are true inclusions, completely closed and delimited by the nuclear membrane, containing degenerate cell organelles and lysosomal proteins. Moreover, their occurrence was positively associated with patient survival but not with tumour grade or stage. In a standardised area a mean of 124 inclusions per specimen was present in the tumorous liver tissue in contrast to 5 inclusions in the non-tumorous adjacent section and 89% of all scrutinised HCC showed at least one membrane-bound nuclear inclusion. Ultrastructural characterisation by transmission electron microscopy revealed degenerative materials such as residues of lysosomes, endoplasmic reticulum and Golgi apparatus within the inclusions. Due to the fact that the content of the inclusions appears to be more condensed than cytoplasm and contains fewer intact cell organelles, we assume that they are not mere invaginations of cytoplasm. Three dimensional (3D) reconstruction of isolated and immunofluorescence stained nuclei showed that the inclusions are completely located within the nucleus without any connection to the cytoplasm. The limiting membrane of the inclusions contained lamin B suggesting nuclear membrane origin. The content of the inclusions stained for the autophagy-associated proteins p62, ubiquitin, LC3B, cathepsin B and cathepsin D. Triple immunofluorescence staining followed by 3D reconstruction revealed co-localisation of p62, ubiquitin and LC3B in the same inclusion. Our observations uncover that these inclusions are real inclusions completely surrounded by the nucleus. We propose that the presence of autophagy-associated proteins and proteases within the inclusions contribute to beneficial survival., (© 2019 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

We examined samples of human pheochromocytoma from 11 patients aged 30-70 years including one case of malignant pheochromocytoma with a view to identifying previously unreported ultrastructural details.We identified two types of nuclear inclusions consisting of irregularly shaped singular or multiple granulofibrillar formations with a typical concentric halo, on the one hand, and accumulations of egg-shaped structures consisting of granules and microfilaments, on the other. In some of the tumor cells, membrane-covered inclusions containing parallel laminar elements arranged in a paracrystalline, periodic fashion, or mega-mitrochondriae characterized by increased electrodensity of their matrix, and fibrillary material in the spaces between the cristae were present. A frequent finding consisted of typical ciliary formations, while rough/smooth tubular aggregates of different size occurred less frequently. Finally, we were able to demonstrate the uptake of norepinephrine by smooth muscle fibers in the periphery of arterial vessels as evidenced by linear accumulations of membrane-covered granules separating bands of contractile smooth muscle components in the peripheral layers of arterial vessels close to norepinephrine producing neoplastic cells.These findings represent ultrastructural features that contribute to further elucidating the ultrastructural characteristics of the human pheochromocytoma.

Phase transitions are ubiquitous in nonliving matter, and recent discoveries have shown that they also play a key role within living cells. Intracellular liquid-liquid phase separation is thought to drive the formation of condensed liquid-like droplets of protein, RNA, and other biomolecules, which form in the absence of a delimiting membrane. Recent studies have elucidated many aspects of the molecular interactions underlying the formation of these remarkable and ubiquitous droplets and the way in which such interactions dictate their material properties, composition, and phase behavior. Here, we review these exciting developments and highlight key remaining challenges, particularly the ability of liquid condensates to both facilitate and respond to biological function and how their metastability may underlie devastating protein aggregation diseases., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Neuronal intranuclear hyaline inclusion disease (NIHID) is a neurodegenerative disorder characterized by the presence of eosinophilic nuclear inclusions (NIs) in diverse cell lines in systemic organs. Adult-onset NIHID typically manifests with dementia associated with leukoencephalopathy. The detection of NIs in skin biopsies is useful for an antemortem diagnosis. A previous analysis suggested that NIs in NIHID originated from nuclear bodies (NBs), an important nuclear domain related to the ubiquitin-p62-mediated protein degradation system. In this study, we analyzed skin samples from 5 NIHID and 5 control cases immunohistochemically and electron microscopically. In the control cases, small but significant amounts of ubiquitin- and p62-positive intranuclear structures were found. These structures were consistently colocalized with promyelocytic leukemia protein (PML), an essential component of NBs, in particular when activated. The p62- and PML-positive structures were more frequently found in NIHID cases. Activated NBs, having a core and a shell, were observed by electron microscopy in control but not in NIHID cases. Instead, immature and mature filamentous NIs were found only in the NIHID cases. Our results indicate that NBs could not be normally activated in the NIHID, and an abnormal alteration of NBs might be related to the pathogenesis of NIHID., (© 2017 American Association of Neuropathologists, Inc. All rights reserved.)

Nuclear glycogen inclusions occur infrequently in pathologic conditions but also in normal human and animal tissues. Their function or significance is unclear. To the best of the authors' knowledge, no reports of nuclear glycogen inclusions in canine parietal cells exist. After initial observations of nuclear inclusions/pseudoinclusions during routine histopathology, the authors retrospectively examined samples of gastric mucosa from dogs presenting with gastrointestinal signs for the presence of intranuclear inclusions/pseudoinclusions and determined their composition using histologic and electron-microscopic methods. In 24 of 108 cases (22%), the authors observed various numbers of intranuclear inclusions/pseudoinclusions within scattered parietal cells. Nuclei were characterized by marked karyomegaly and chromatin margination around a central optically empty or slightly eosinophilic area. The intranuclear inclusions/pseudoinclusions stained positive with periodic acid-Schiff (PAS) and were diastase sensitive, consistent with glycogen. Several PAS-positive/diastase-sensitive sections were further examined by transmission electron microscopy, also using periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining to identify polysaccharides. Ultrastructurally, the nuclear inclusions were composed of electron-dense particles that were not membrane bound, without evidence of nuclear membrane invaginations or cytoplasmic organelles in the nuclei, and positive staining with PA-TCH-SP, confirming a glycogen composition. No cytoplasmic glycogen deposits were observed, suggesting that the intranuclear glycogen inclusions were probably synthesized in loco. Nuclear glycogen inclusions were not associated with gastritis or colonization by Helicobacter-like organisms ( P > .05). Our findings suggest that nuclear glycogen inclusions in canine parietal cells could be an incidental finding. Nevertheless, since nuclear glycogen is present in several pathologic conditions, further investigations could be warranted to determine their true significance.

The nuclear body paraspeckle is built on the lncRNA Neat1 and plays important roles in gene regulation. In this issue, West et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601071) use super-resolution structured illumination microscopy to show that paraspeckles are organized in a core-shell spheroidal structure composed of Neat1 and seven proteins., (© 2016 Hu et al.)

The ultrastructure of the retinal pigment epithelium of a diurnal rodent (Brandt's vole) was described taking into account 1) the functions of the pigment epithelium as a participant in the renewal of photoreceptor outer segment and. 2) digestion of outer segment membranes into phagosomes of the retinal pigment epithelium. The myeloid bodies were observed after exposure of the pigment epithelium to light (200 lux, 4 hours) and darkness (0,1 lux, 1,5-hour). In the cytoplasm of the pigment epithelium of the vole no myeloid bodies were observed. Instead of it small lamellar bodies, which have the spiral form and size (from - 200 to 400 nm) were found. The structure of these lamellar bodies was described. Furthermore, the structures, which were presumably responsible for the transport of the digested material, were revealed. The evidence of it is the presence of 1) dense precipitate in the apical domain of the pigment epithelium and 2) microtubules which participate in transport of this precipitate.

The sizes of nuclear bodies and other nuclear structures are normally no more than a few hundred nanometers. This size is below the resolution limit of light microscopy and thus requires electron microscopy for direct observation. Recent developments in super-resolution microscopy have extended the resolution of light microscopy to beyond 100 nm. Here, we describe a super-resolution technique, gated STED, for the analysis of the structure of nuclear bodies, with emphasis on the sample preparation and other technical tips that are important to obtain high-quality super-resolution images.

Recent immunofluorescent (IF) studies have discovered a variety of nuclear foci that have no known ultrastructurally defined counterpart. Using antibodies as ligands, immuno-electron microscopy (I-EM) is the method of choice for high-resolution recognition of these newly described nuclear compartments. However, noncoding RNAs (ncRNAs) have also been shown to be frequent components, sometimes essential, of nuclear bodies so that electron microscopic in situ hybridization (EM-ISH) can be used as an alternative means to characterize nuclear foci at the EM level. Among the array of protocols available, Lowicryl embedding of chemically fixed cells allows for high preservation of both nuclear structures and antigenicity and provides stable cell and tissue samples that can be re-probed whenever new antibodies or probes become available. Rapid and robust protocols are available for both I-EM and EM-ISH post-embedding techniques so that they can be combined on the same sections, providing ultrastructural and molecular insights into newly "emerging" nuclear bodies.

Fluorescence microscopy is a powerful technique that has become central in the study of the structure and function of biological specimens. This is due in large part to its specificity and versatility. Although an understanding of structure-typically through high-resolution imaging of fixed material-has proved an important tool to understanding function, fluorescence microscopy also offers a mechanism to interrogate cells in the living state, providing a means to explore dynamic process within the specimen over long time periods at high temporal resolution. The cell nucleus is a highly compartmented environment whose components are often highly motile and in a constant state of flux. The ability to monitor the dynamic behavior of nuclear bodies by live-cell imaging provides the researcher with important information regarding underlying mechanistic processes relating to their formation and maintenance. Two techniques have proved particularly valuable to our study of cellular dynamics and molecular mobility, namely, time-lapse imaging and fluorescence recovery after photobleaching (FRAP). Time-lapse microscopy allows for qualitative and quantitative analysis of a wide range of events at the cellular and subcellular level. FRAP provides a mechanism to study the mobility of a population of proteins in a range of conditions within discrete areas of the biological specimen. Therefore, fluorescence microscopy is unique in its ability to provide data at high temporal resolution and in such exquisite detail.

Fluorescence microscopy (FM) and electron microscopy (EM) are powerful tools for observing molecular components in cells. FM can provide temporal information about cellular proteins and structures in living cells. EM provides nanometer resolution images of cellular structures in fixed cells. We have combined FM and EM to develop a new method of correlative light and electron microscopy (CLEM), called "Live CLEM." In this method, the dynamic behavior of specific molecules of interest is first observed in living cells using fluorescence microscopy (FM) and then cellular structures in the same cell are observed using electron microscopy (EM). Following image acquisition, FM and EM images are compared to enable the fluorescent images to be correlated with the high-resolution images of cellular structures obtained using EM. As this method enables analysis of dynamic events involving specific molecules of interest in the context of specific cellular structures at high resolution, it is useful for the study of nuclear structures including nuclear bodies. Here we describe Live CLEM that can be applied to the study of nuclear structures in mammalian cells.

In order to gain insights on the nuclear organization in mammalian cells, we characterized ultrastructurally nuclear bodies (NBs) previously described as fluorescent foci. Using high resolution immunoelectron microscopy (I-EM), we provide evidence that CNoBs (CRM1-Nucleolar bodies) and INBs (Intranucleolar bodies) are distinct genuine nucleolar structures in untreated HeLa cells. INBs are fibrillar and concentrate the post-translational modifiers SUMO1 and SUMO-2/3 as strongly as PML bodies. In contrast, the smallest CRM1-labeled CNoBs are vitreous, preferentially located at the periphery of the nucleolus and, intricately linked to the chromatin network. Upon blockage of the CRM1-dependent nuclear export by leptomycin B (LMB), CNoBs disappear while p62/SQSTM1-containing fibrillar nuclear bodies are induced. These p62 bodies are enriched in ubiquitinated proteins. They progressively associate with PML bodies to form hybrid bodies of which PML decorates the periphery while p62/SQSTM1 is centrally-located. Our study is expanding the repertoire of nuclear bodies; revealing a previously unrecognized composite nucleolar landscape and a new mode of interactions between ubiquitous (PML) and stress-induced (p62) nuclear bodies, resulting in the formation of hybrid bodies.

Epidemiologic and experimental data suggest the involvement of cholesterol metabolism in the development and progression of Alzheimer disease and Niemann-Pick type C disease, but not of frontotemporal dementias. In these 3 neurodegenerative diseases, however, protein tau hyperphosphorylation and aggregation into neurofibrillary tangles are observed. To elucidate the relationship between cholesterol and tau, we compared sterol levels of neurons burdened with neurofibrillary tangles with those of their unaffected neighbors using semiquantitative filipin fluorescence microscopy in mice expressing P301L mutant human tau (a well-described model of FTDP-17) and in P301L transgenic mice lacking apolipoprotein E (the major cholesterol transporter in the brain). Cellular unesterified cholesterol was higher in neurons affected by tau pathology irrespective of apolipoprotein E deficiency. This argues for an impact of tau pathology on cellular cholesterol homeostasis. We suggest that there is a bidirectional mode of action: Disturbances in cellular cholesterol metabolism may promote tau pathology, but tau pathology may also alter neuronal cholesterol homeostasis; once it is established, a vicious cycle may promote neurofibrillary tangle formation.

In progressive multifocal leukoencephalopathy, JC virus-infected oligodendroglia display 2 distinct patterns of intranuclear viral inclusions: full inclusions in which progeny virions are present throughout enlarged nuclei and dot-shaped inclusions in which virions are clustered in subnuclear domains termed "promyelocytic leukemia nuclear bodies" (PML-NBs). Promyelocytic leukemia nuclear bodies may serve a scaffolding role in viral progeny production. We analyzed the formation process of intranuclear viral inclusions by morphometry and assessed PML-NB alterations in the brains of 2 patients with progressive multifocal leukoencephalopathy. By immunohistochemistry, proliferating cell nuclear antigen was most frequently detected in smaller nuclei; cyclin A was detected in larger nuclei. This suggests an S-to-G2 cell cycle transition in infected cells associated with nuclear enlargement. Sizes of PML-NBs were variable, but they were usually either small speckles 200 to 400 nm in diameter or distinct spherical shells with a diameter of 1 μm or more. By confocal microscopy, JC virus capsid proteins were associated with both small and large PML-NBs, but disruption of large PML-NBs was observed by ground-state depletion fluorescence nanoscopy. Clusters of progeny virions were also detected by electron microscopy. Our data suggest that, in progressive multifocal leukoencephalopathy, JC virus produces progeny virions in enlarging oligodendrocyte nuclei in association with growing PML-NBs and with cell cycle transition through an S-to-G2-like state.

The well-known debate on the nature and origin of intracellular inclusions (ICIs) in silicified microfossils from the early Neoproterozoic Bitter Springs Formation has recently been revived by reports of possible fossilized nuclei in phosphatized animal embryo-like fossils from the Ediacaran Doushantuo Formation of South China. The revisitation of this discussion prompted a critical and comprehensive investigation of ICIs in some of the oldest indisputable eukaryote microfossils-the ornamented acritarchs Dictyosphaera delicata and Shuiyousphaeridium macroreticulatum from the Paleoproterozoic Ruyang Group of North China-using a suite of characterization approaches: scanning electron microscopy (SEM), transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM). Although the Ruyang acritarchs must have had nuclei when alive, our data suggest that their ICIs represent neither fossilized nuclei nor taphonomically condensed cytoplasm. We instead propose that these ICIs likely represent biologically contracted and consolidated eukaryotic protoplasts (the combination of the nucleus, surrounding cytoplasm, and plasma membrane). As opposed to degradational contraction of prokaryotic cells within a mucoidal sheath-a model proposed to explain the Bitter Springs ICIs-our model implies that protoplast condensation in the Ruyang acritarchs was an in vivo biologically programmed response to adverse conditions in preparation for encystment. While the discovery of bona fide nuclei in Paleoproterozoic acritarchs would be a substantial landmark in our understanding of eukaryote evolution, the various processes (such as degradational and biological condensation of protoplasts) capable of producing nuclei-mimicking structures require that interpretation of ICIs as fossilized nuclei be based on comprehensive investigations., (© 2013 John Wiley & Sons Ltd.)

Nuclear inclusion or pseudoinclusion is a peculiar cytological feature, and its recognition in appropriate clinicopathological settings can aid in the diagnosis of several disease entities. To the best of our knowledge, only 1 case of pulmonary neuroendocrine tumor (NET) with nuclear pseudoinclusion has been reported. A review of 227 patients who had undergone surgical resection for pulmonary NETs revealed 2 tumors with different mechanisms of nuclear inclusion. To explore the cause of nuclear inclusion, NET with nuclear inclusion was characterized immunohistochemically and ultrastructurally. Nuclear inclusions were observed in 2 of the 227 (0.9%) patients with pulmonary NETs. The first patient was a 46-year-old woman with small cell carcinoma. Tumor cells with nuclear inclusions were distributed focally. Ultrastructural analysis showed that these inclusions were pseudoinclusions. The second patient was a 62-year-old man with large-cell neuroendocrine carcinoma. Nuclear inclusions were observed in the focal area of the tumor. Immunohistochemical analysis revealed that the intra-nuclear materials consisted of biotin and aberrant cytoplasmic and nuclear accumulation of β-catenin. Mutational analysis revealed a CTNNB1 gene mutation. Although very rare, diagnostic errors may be observed in cases of pulmonary NETs with nuclear inclusions. The mechanisms of nuclear inclusion differed, with one due to herniation of the cytoplasm into the nucleus (pseudoinclusion) and the other due to accumulation of biotin resulting from a CTNNB1 gene mutation., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

ORF78 (ac78) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a baculovirus core gene of unknown function. To determine the role of ac78 in the baculovirus life cycle, an AcMNPV mutant with ac78 deleted, Ac78KO, was constructed. Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. After transfection into Spodoptera frugiperda cells, Ac78KO produced a single-cell infection phenotype, indicating that no infectious budded viruses (BVs) were produced. The defect in BV production was also confirmed by both viral titration and Western blotting. However, viral DNA replication was unaffected, and occlusion bodies were formed. An analysis of BVs and occlusion-derived viruses (ODVs) revealed that AC78 is associated with both forms of the virions and is an envelope structural protein. Electron microscopy revealed that AC78 also plays an important role in the embedding of ODV into the occlusion body. The results of this study demonstrate that AC78 is a late virion-associated protein and is essential for the viral life cycle.

Important insights into nuclear function would arise if gene loci physically interacting with particular subnuclear domains could be readily identified. Immunofluorescence microscopy combined with fluorescence in situ hybridization (immuno-FISH), the method that would typically be used in such a study, is limited by spatial resolution and requires prior assumptions for selecting genes to probe. Our new technique, immuno-TRAP, overcomes these limitations. Using promyelocytic leukemia nuclear bodies (PML NBs) as a model, we used immuno-TRAP to determine if specific genes localize within molecular dimensions with these bodies. Although we confirmed a TP53 gene-PML NB association, immuno-TRAP allowed us to uncover novel locus-PML NB associations, including the ABCA7 and TFF1 loci and, most surprisingly, the PML locus itself. These associations were cell type specific and reflected the cell's physiological state. Combined with microarrays or deep sequencing, immuno-TRAP provides powerful opportunities for identifying gene locus associations with potentially any nuclear subcompartment.

We used hybrid detectors (HyDs) to monitor the trajectories and interactions of promyelocytic leukemia (GFP-PML) nuclear bodies (NBs) and mCherry-53BP1-positive DNA lesions. 53BP1 protein accumulates in NBs that occur spontaneously in the genome or in γ-irradiation-induced foci. When we induced local DNA damage by ultraviolet irradiation, we also observed accumulation of 53BP1 proteins into discrete bodies, instead of the expected dispersed pattern. In comparison with photomultiplier tubes, which are used for standard analysis by confocal laser scanning microscopy, HyDs significantly eliminated photobleaching of GFP and mCherry fluorochromes during image acquisition. The low laser intensities used for HyD-based confocal analysis enabled us to observe NBs for the longer time periods, necessary for studies of the trajectories and interactions of PML and 53BP1 NBs. To further characterize protein interactions, we used resonance scanning and a novel bioinformatics approach to register and analyze the movements of individual PML and 53BP1 NBs. The combination of improved HyD-based confocal microscopy with a tailored bioinformatics approach enabled us to reveal damage-specific properties of PML and 53BP1 NBs.

Late infantile neuronal ceroid lipofuscinosis (Jansky-Bielchowsky disease) is a rare disease caused by mutations in the CLN2 gene. The authors report the clinical outcome and correlate with genotype in 12 Spanish patients with this disease. Psychomotor regression, epilepsy, and other clinical symptoms/signs were assessed. Age at onset of clinical symptoms ranged from 18 months to 3.7 years, and they included delayed speech and simple febrile seizures followed by epilepsy. Partial seizures and myoclonic jerks occurred at an earlier age (median 3.4 and 3.7 years, respectively) than ataxia and cognitive decline (median 4 years). Clinical regression was initiated by loss of sentences (median 3.7 years) followed by loss of walking ability and absence of language (median 4.5 years). Patients showed blindness and lost sitting ability at similar age (median 5 years). The authors report 4 novel mutations in the CLN2 gene. This study provides detailed information about the natural history of this disease.

The cell nucleus contains many distinct subnuclear compartments, domains, and bodies that vary in their composition, structure, and function. While the cellular constituents that occupy the subnuclear regions may be well known, defining the structural details of the molecular assembly of the constituents has been more difficult. A correlative fluorescence and energy-filtering transmission electron microscopy (EFTEM) imaging method has the ability to provide these details. The correlative microscopy method described here allows the tracking of subnuclear structures from specific cells by fluorescence microscopy and then, using electron energy loss imaging in the transmission electron microscope, reveals the ultrastructural features of the nuclear components along with endogenous elemental information that relates directly to the biochemical composition of the structure. The ultrastructural features and composition of well-characterized PML bodies and interchromatin granule clusters are compared to those of ligand-activated glucocorticoid receptor (GR) foci, with GR foci containing fibrogranular nucleic acid-containing features and PML bodies being devoid of nucleic acid.

The nucleus of higher eukaryotes, such as humans and mice, is compartmentalized into multiple nuclear bodies, an organization that allows for the regulation of complex gene expression pathways that are characteristic of these organisms. Paraspeckles are recently discovered, mammalian-specific nuclear bodies built on a long, non-protein-coding RNA, NEAT1 (nuclear-enriched abundant transcript 1), which assembles various protein components including RNA-binding proteins of the DBHS (Drosophila behavior and human splicing) family. Paraspeckles have been proposed to control several biological processes, such as stress responses and cellular differentiation, but their function at the whole animal level remains unclear. In this review, we summarize a series of studies on paraspeckles that have been carried out in the decade since their discovery and discuss their physiological function and molecular mechanism.

Intranuclear pseudoinclusions represent invaginations of the cytoplasm into the nucleus. The nuclear pseudoinclusions are generally easier to appreciate in cytological preparations than frozen sections or histopathology thus forming an important cytological feature. In the present article we have reviewed the morphology, pathogenesis, differential diagnosis, and clinical significance of intranuclear cytoplasmic pseudoinclusion., (Copyright © 2011 Wiley Periodicals, Inc.)

Background Information: Promyelocytic leukaemia (PML) bodies are specific nuclear structures with functional significance for acute promyelocytic leukaemia. In this study, we analysed the trajectories of PML bodies using single-particle tracking., Results: We observed that the recovery of PML protein after photobleaching was ATP dependent in both wild-type (wt) and A-type lamin-deficient cells. The movement of PML bodies was faster and the nuclear area occupied by particular PML bodies was larger in A-type lamin-deficient fibroblasts compared with their wt counterparts. Moreover, dysfunction of the LMNA gene increased the frequency of mutual interactions between individual PML bodies and influenced the morphology of these domains at the ultrastructural level. As a consequence of A-type lamin deficiency, PML protein accumulated in nuclear blebs and frequently appeared at the nuclear periphery., Conclusions: We suggest that the physiological function of lamin A proteins is important for events that occur in the compartment of PML bodies. This observation was confirmed in other experimental models characterised by lamin changes, including apoptosis or the differentiation of mouse embryonic stem cells., (Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.)

Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder caused by JC virus infection. JC virus was recently found to target promyelocytic leukemia nuclear bodies (PML-NBs), punctuate domains in the nuclei. Thus, the virus progenies cluster in dots as intranuclear inclusions (ie, as dot-shaped inclusions). In the present study, both the viral major and minor capsid proteins were expressed from polycistronic expression vectors with a powerful promoter, and formation into virus-like particles (VLPs) was examined by electron microscopy. When the upstream regulatory sequence including the agnogene (nt 275 to 490) was present, capsid protein expression was suppressed, but numerous VLPs were efficiently formed with restricted accumulation to PML-NBs. VLPs were uniform, and the cells were severely degraded. In contrast, when the 5' terminus of the agnogene (nt 275 to 409; 135 bp) was deleted, capsid protein expression was markedly enhanced, but VLPs were more randomly produced in the nucleus outside of PML-NBs. VLPs were pleomorphic, and cell degradation was minimal. JC virus association with PML-NBs was confirmed in human brain tissues by structured illumination microscopy. PML-NBs were shaped in spherical shells, with viral capsid proteins circumscribing the surface. These findings indicate that PML-NBs are intranuclear locations for pathogenic JC virus proliferation. Either the agnogene or its product likely supports efficient progeny production at PML-NBs, leading to subsequent degeneration of host glial cells., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Recently, we reported widespread intraneuronal prion protein (PrP) immunoreactivity in genetic Creutzfeldt-Jakob disease (CJD) associated with the E200K mutation. Here, we evaluated 6 cases ofsporadic CJD MM type 1, 5 MV type 2, and 7 VV type 2 and compared their anatomical appearance with that of 29 E200K genetic CJD (gCJD) cases. We also performed double immunolabeling for ubiquitin, p62, early endosomal marker rab5, and immunogold electronmicroscopy in 3 cases. We identified 4 morphological types of intraneuronal PrP immunoreactivity: one type, defined as multiple globular structures, was significantly associated with a subset of E200K gCJD cases and was distinct from the intraneuronal small dotlike PrP immunoreactivity seen in sporadic CJD. Whereas the latter colocalized with rab5, there were single large (7.5 μm-15 μm) globular inclusion body-like structures detected predominantly but not exclusively in E200K gCJD; these were immunoreactive in part for ubiquitin and p62 and showed focal γ-tubulin immunoreactivity, suggesting aggresome features. Ultrastructural examination using immunogold revealed PrP localization in aggresome-like structures and in autophagic vacuoles. These findings suggest that the permanent production of mutant PrP in the E200K gCJD cases overwhelms the ubiquitin-proteasome system and shifts the balance toward selectivemacroautophagy and/or to ubiquitinated inclusion body and aggresome formation as a cytoprotective effort to sequester the mutant protein.

α-Synuclein and mutant huntingtin are the major constituents of the intracellular aggregates that characterize the pathology of Parkinson's disease (PD) and Huntington's disease (HD), respectively. α-Synuclein is likely to be a major contributor to PD, since overexpression of this protein resulting from genetic triplication is sufficient to cause human forms of PD. We have previously demonstrated that wild-type α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Overexpression of human wild-type α-synuclein in cells and Drosophila models of HD worsens the disease phenotype. Here, we examined whether α-synuclein overexpression also worsens the HD phenotype in a mammalian system using two widely used N-terminal HD mouse models (R6/1 and N171-82Q). We also tested the effects of α-synuclein deletion in the same N-terminal HD mouse models, as well as assessed the effects of α-synuclein deletion on macroautophagy in mouse brains. We show that overexpression of wild-type α-synuclein in both mouse models of HD enhances the onset of tremors and has some influence on the rate of weight loss. On the other hand, α-synuclein deletion in both HD models increases autophagosome numbers and this is associated with a delayed onset of tremors and weight loss, two of the most prominent endophenotypes of the HD-like disease in mice. We have therefore established a functional link between these two aggregate-prone proteins in mammals and provide further support for the model that wild-type α-synuclein negatively regulates autophagy even at physiological levels.

To analyze the subcellular trafficking of herpesvirus capsids, the small capsid protein has been labeled with different fluorescent proteins. Here, we analyzed the infectivity of several HSV1(17(+)) strains in which the N-terminal region of the non-essential small capsid protein VP26 had been tagged at different positions. While some variants replicated with similar kinetics as their parental wild type strain, others were not infectious at all. Improper tagging resulted in the aggregation of VP26 in the nucleus, prevented efficient nuclear egress of viral capsids, and thus virion formation. Correlative fluorescence and electron microscopy showed that these aggregates had sequestered several other viral proteins, but often did not contain viral capsids. The propensity for aggregate formation was influenced by the type of the fluorescent protein domain, the position of the inserted tag, the cell type, and the progression of infection. Among the tags that we have tested, mRFPVP26 had the lowest tendency to induce nuclear aggregates, and showed the least reduction in replication when compared to wild type. Our data suggest that bona fide monomeric fluorescent protein tags have less impact on proper assembly of HSV1 capsids and nuclear capsid egress than tags that tend to dimerize. Small chemical compounds capable of inducing aggregate formation of VP26 may lead to new antiviral drugs against HSV infections.

Aim: To document and clarify the nature of intranuclear inclusions of luminal epithelium in benign proliferative breast lesions., Methods and Results: Five benign breast lesions were selected which showed intranuclear inclusions within epithelial cells on light microscopy. Following confirmation of their luminal epithelial (non-myoepithelial) localisation by immunohistochemistry, ultrastructural examination was performed with the following observations: (1) presence of deep nuclear indentations occasionally verging on nuclear inclusions; (2) inclusions with features of helioid bodies; and (3) a morphological spectrum of helioid bodies and their focal coexistence., Conclusion: Intranuclear inclusions of breast epithelium are likely of cytoplasmic origin. Helioid bodies may be formed by a stepwise process, the nature of which needs further study.

Context: Recently we have observed distinctive acidophilic intranuclear inclusions in cases of usual intraductal hyperplasia of the breast. Similar inclusions were described almost 20 years ago in cases of mammary hyperplasia. These correlated ultrastructurally with so-called helioid inclusions. However, there since has been little discussion of these inclusions in the literature., Objective: To examine the incidence and specificity of these inclusions in proliferative lesions of the breast., Design: Forty cases of usual intraductal hyperplasia, 15 cases of atypical ductal hyperplasia, and 34 cases of low-grade ductal carcinoma in situ were examined for the presence of acidophilic intranuclear inclusions., Results: Acidophilic intranuclear inclusions were present in 50% of cases of usual intraductal hyperplasia (20 of 40) but were not identified in any cases of atypical ductal hyperplasia (0 of 15) or low-grade ductal carcinoma in situ (0 of 34)., Conclusions: Acidophilic intranuclear inclusions appear to be a common, specific feature found in usual intraductal hyperplasia and may be helpful in distinguishing it from atypical ductal hyperplasia and low-grade ductal carcinoma in situ in some cases. Elucidating the nature of these inclusions may provide insight into the pathogenesis of usual intraductal hyperplasia.

Background: Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. Because of the variety of clinical manifestations, antemortem diagnosis of NIID is difficult., Methods: Seven skin biopsy samples from patients with familial NIID were evaluated histochemically, and the results were compared with those of skin samples from normal control subjects and from patients with other neurologic diseases. We also examined skin biopsy samples from patients with NIID by electron microscopy., Results: In NIID skin biopsy samples, intranuclear inclusions were observed in adipocytes, fibroblasts, and sweat gland cells. These inclusions were stained with both anti-ubiquitin and anti-SUMO1 antibodies. Electron microscopy revealed that the features of the intranuclear inclusions in adipocytes, fibroblasts, and sweat gland cells were identical to those of neuronal cells. Approximately 10% of adipocytes showed intranuclear inclusions. No intranuclear inclusions were identified in the skin samples from normal control subjects and patients with other neurologic diseases., Conclusions: Skin biopsy is an effective and less invasive antemortem diagnostic tool for NIID.

We report an incipient case of intranuclear inclusion body disease (INIBD) in a 78-year-old woman. No apparent neurological symptoms were noticed during the clinical course. Post mortem examination revealed widespread occurrence of eosinophilic intranuclear inclusions in neuronal and glial cells of the central and peripheral nervous systems, as well as in parenchymal cells of the visceral organs. The inclusions were observed more frequently in glial cells than in neuronal cells. Ultrastructurally, the inclusions consisted of granular and filamentous material. Immunohistochemically, the inclusions were positive for ubiquitin, ubiquitin-related proteins (NEDD8 ultimate buster 1, small ubiquitin modifier-1, small ubiquitin modifier-2 and p62), promyelocytic leukemia protein and abnormally expanded polyglutamine. Consistent with previous studies, the vast majority of inclusion-bearing glial cells were astrocytes. Furthermore, p25α-positive oligodendrocytes rarely contained intranuclear inclusions. These findings suggest that INIBD may occur in non-demented elderly individuals and that oligodendrocyte is also involved in the disease process of INIBD., (© 2010 Japanese Society of Neuropathology.)

Many models of human tauopathies have been generated in mice by expression of a human mutant tau with maintained expression of mouse endogenous tau. Because murine tau might interfere with the toxic effects of human mutant tau, we generated a model in which a pathogenic human tau protein is expressed in the absence of wild-type tau protein, with the aim of facilitating the study of the pathogenic role of the mutant tau and to reproduce more faithfully a human tauopathy. The Tg30 line is a tau transgenic mouse model overexpressing human 1N4R double-mutant tau (P301S and G272V) that develops Alzheimer's disease-like neurofibrillary tangles in an age-dependent manner. By crossing Tg30 mice with mice invalidated for their endogenous tau gene, we obtained Tg30xtau(-/-) mice that express only exogenous human double-mutant 1N4R tau. Although Tg30xtau(-/-) mice express less tau protein compared with Tg30, they exhibit signs of decreased survival, increased proportion of sarkosyl-insoluble tau in the brain and in the spinal cord, increased number of Gallyas-positive neurofibrillary tangles in the hippocampus, increased number of inclusions in the spinal cord, and a more severe motor phenotype. Deletion of murine tau accelerated tau aggregation during aging of this mutant tau transgenic model, suggesting that murine tau could interfere with the development of tau pathology in transgenic models of human tauopathies., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

We describe a severe congenital myopathy patient of Xhosa native African origin with a novel de novo p.Gly152Ala skeletal muscle α-actin gene (ACTA1) mutation, who died at 6 months of age. The muscle pathology demonstrated abundant cytoplasmic and intranuclear rods, core-like areas and the unusual feature of larger type I than type II fibres. Our results further expand the phenotypes associated with ACTA1 mutations and provide support for the hypothesis that the structural abnormalities seen are a pathological continuum dependent on the precise mutation and biopsy location. Our results also demonstrate the likely world-wide distribution of de novo mutations in this gene., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

Giant cell angiofibroma was first described as a distinctive orbital soft-tissue tumour in male adults; it is now recognized that this mesenchymal tumour can present in other anatomical regions. In this article, a case of giant cell angiofibroma of parapharyngeal space in a 25-year-old woman is described. Clinicopathologic features of this tumour are reviewed. To the authors' knowledge, this is the first reported case of giant cell angiofibroma arising in the parapharyngeal space., (Copyright © 2010 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Huntington disease (HD) is a fatal neurodegenerative disease with no effective treatment. In the R6/1 mouse model of HD, environmental enrichment delays the neurologic phenotype onset and prevents cerebral volume loss by unknown molecular mechanisms. We examined the effects of environmental enrichment on well-characterized neuropathological parameters in a mouse model of HD. We found a trend toward preservation of downregulated neurotransmitter receptors in striatum of environmentally enriched mice and assessed possible enrichment-related modifications in gene expression using microarrays. We observed similar gene expression changes in R6/1 and R6/2 transgenic mice but found no specific changes in enrichment-related microarray expression profiles in either transgenic or wild-type mice. Furthermore, specific corrections in transprotein-induced transcriptional dysregulation in R6/1 mice were not detected by microarray profiling. However, gene-specific analyses suggested that long-term environmental enrichment may beneficially modulate gene expression dysregulation. Finally, environmental enrichment significantly decreased neuronal intranuclear inclusion load, despite unaffected transgene expression levels. Thus, the therapeutic effects of environmental enrichment likely contribute to decreasing aggregated polyglutamine protein levels without exerting strong effects on gene expression.

Promyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50- to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PML-NBs serves to concentrate different biological activities while allowing for an efficient exchange of components.

REGgamma (also called PA28gamma or PSME3) is a proteasome activator involved in the degradation of several proteins that regulate cell cycle and transcription. Recently, we demonstrated that this protein has a role also in the maintenance of chromosomal stability and in the response to spindle damaging agents. Here we report for the first time that REGgamma interacts with the promyelocytic leukemia protein (PML), accumulates in PML nuclear bodies (PML-NBs), but it does not play any role in normal or arsenic-induced PML degradation. However, REGgamma seems to regulate PML-NBs number, since its deficiency causes an increase in PML-NBs, which can be overcome by increased levels of SUMO1, and its overexpression has the opposite effect. We additionally found that REGgamma interacts with the DNA damage checkpoint kinase Chk2, whose presence is necessary for the increase of PML-NBs induced by REGgamma deficiency, and that REGgamma depletion resulted in a partial restoration of PML-NBs in APL derived cells. Altogether, these results underline a new role for REGgamma in the control and regulation of PML subnuclear structures.

Huntington's disease (HD) is caused by an expanded CAG repeat in the HD gene. The pathological threshold for expansion in HD is around 36 CAG repeats, although 'super-long' expansions are found in brains of HD patients. We examined the effect of varying the CAG repeat length (from 170 to 450) on behavior and neuropathology of R6/2 mice. Unexpectedly, we found that increasing the repeat length delayed onset of disease and prolonged survival, from around 4 months to over 18 months in mice with the longest repeats. The delay in onset correlated with a delayed appearance of neuronal intranuclear inclusions (NIIs). However, super-long CAG repeats are not neuroprotective. Mice carrying 2 copies of the mutant transgene die earlier than those carrying a single copy. Furthermore, neurodegeneration is present in super-long repeat length mice at mid-stage disease, whereas little neurodegeneration is seen in mice with shorter CAG repeats until end stage. Expanding the CAG repeat beyond the range where NII formation is the dominant pathology has unmasked a slowly progressing neurological phenotype in R6/2 mice with brain pathology, including the identification of a novel form of inclusion, that more closely resembles that seen in adult onset cases of HD. This mouse may represent a better model for adult-onset HD than R6/2 mice with shorter repeats.