The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation.

Microbes or danger signals trigger inflammasome sensors, which induce polymerization of the adaptor ASC and the assembly of ASC specks. ASC specks recruit and activate caspase-1, which induces maturation of the cytokine interleukin 1ß (IL-1ß) and pyroptotic cell death. Here we found that after pyroptosis, ASC specks accumulated in the extracellular space, where they promoted further maturation of IL-1ß. In addition, phagocytosis of ASC specks by macrophages induced lysosomal damage and nucleation of soluble ASC, as well as activation of IL-1ß in recipient cells. ASC specks appeared in bodily fluids from inflamed tissues, and autoantibodies to ASC specks developed in patients and mice with autoimmune pathologies. Together these findings reveal extracellular functions of ASC specks and a previously unknown form of cell-to-cell communication.

FTLD-TDP assemblies seed neoaggregates with subtype-specific features via a prion-like cascade.

Morphologically distinct TDP-43 aggregates occur in clinically different FTLD-TDP subtypes, yet the mechanism of their emergence and contribution to clinical heterogeneity are poorly understood. Several lines of evidence suggest that pathological TDP-43 follows a prion-like cascade, but the molecular determinants of this process remain unknown. We use advanced microscopy techniques to compare the seeding properties of pathological FTLD-TDP-A and FTLD-TDP-C aggregates. Upon inoculation of patient-derived aggregates in cells, FTLD-TDP-A seeds amplify in a template-dependent fashion, triggering neoaggregation more efficiently than those extracted from FTLD-TDP-C patients, correlating with the respective disease progression rates. Neoaggregates are sequentially phosphorylated with N-to-C directionality and with subtype-specific timelines. The resulting FTLD-TDP-A neoaggregates are large and contain densely packed fibrils, reminiscent of the pure compacted fibrils present within cytoplasmic inclusions in postmortem brains. In contrast, FTLD-TDP-C dystrophic neurites show less dense fibrils mixed with cellular components, and their respective neoaggregates are small, amorphous protein accumulations. These cellular seeding models replicate aspects of the patient pathological diversity and will be a useful tool in the quest for subtype-specific therapeutics.

Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction.

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.

Tanycytes and a differential fatty acid metabolism in the hypothalamus.

Although the brain controls all main metabolic pathways in the whole organism, its lipid metabolism is partially separated from the rest of the body. Circulating lipids and other metabolites are taken up into brain areas like the hypothalamus and are locally metabolized and sensed involving several hypothalamic cell types. In this study we show that saturated and unsaturated fatty acids are differentially processed in the murine hypothalamus. The observed differences involve both lipid distribution and metabolism. Key findings were: (i) hypothalamic astrocytes are targeted by unsaturated, but not saturated lipids in lean mice; (ii) in obese mice labeling of these astrocytes by unsaturated oleic acid cannot be detected unless ß-oxidation or ketogenesis is inhibited; (iii) the hypothalamus of obese animals increases ketone body and neutral lipid synthesis while tanycytes, hypothalamic cells facing the ventricle, increase their lipid droplet content; and (iv) tanycytes show different labeling for saturated or unsaturated lipids. Our data support a metabolic connection between tanycytes and astrocytes likely to impact hypothalamic lipid sensing. GLIA 2017;65:231-249.

CRIS-a novel cAMP-binding protein controlling spermiogenesis and the development of flagellar bending.

The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca(2+) regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca(2+)-regulated proteins that--in mature sperm--are involved in flagellar bending.

The three-dimensional molecular structure of the desmosomal plaque.

The cytoplasmic surface of intercellular junctions is a complex network of molecular interactions that link the extracellular region of the desmosomal cadherins with the cytoskeletal intermediate filaments. Although 3D structures of the major plaque components are known, the overall architecture remains unknown. We used cryoelectron tomography of vitreous sections from human epidermis to record 3D images of desmosomes in vivo and in situ at molecular resolution. Our results show that the architecture of the cytoplasmic surface of the desmosome is a 2D interconnected quasiperiodic lattice, with a similar spatial organization to the extracellular side. Subtomogram averaging of the plaque region reveals two distinct layers of the desmosomal plaque: a low-density layer closer to the membrane and a high-density layer further away from the membrane. When combined with a heuristic, allowing simultaneous constrained fitting of the high-resolution structures of the major plaque proteins (desmoplakin, plakophilin, and plakoglobin), it reveals their mutual molecular interactions and explains their stoichiometry. The arrangement suggests that alternate plakoglobin-desmoplakin complexes create a template on which desmosomal cadherins cluster before they stabilize extracellularly by binding at their N-terminal tips. Plakophilins are added as a molecular reinforcement to fill the gap between the formed plaque complexes and the plasma membrane.

The molecular architecture of cadherins in native epidermal desmosomes.

Desmosomes are cadherin-based adhesive intercellular junctions, which are present in tissues such as heart and skin. Despite considerable efforts, the molecular interfaces that mediate adhesion remain obscure. Here we apply cryo-electron tomography of vitreous sections from human epidermis to visualize the three-dimensional molecular architecture of desmosomal cadherins at close-to-native conditions. The three-dimensional reconstructions show a regular array of densities at approximately 70 A intervals along the midline, with a curved shape resembling the X-ray structure of C-cadherin, a representative 'classical' cadherin. Model-independent three-dimensional image processing of extracted sub-tomograms reveals the cadherin organization. After fitting the C-cadherin atomic structure into the averaged sub-tomograms, we see a periodic arrangement of a trans W-like and a cis V-like interaction corresponding to molecules from opposing membranes and the same cell membrane, respectively. The resulting model of cadherin organization explains existing two-dimensional data and yields insights into a possible mechanism of cadherin-based cell adhesion.

Alignator: a GPU powered software package for robust fiducial-less alignment of cryo tilt-series.

The robust alignment of tilt-series collected for cryo-electron tomography in the absence of fiducial markers, is a problem that, especially for tilt-series of vitreous sections, still represents a significant challenge. Here we present a complete software package that implements a cross-correlation-based procedure that tracks similar image features that are present in several micrographs and explores them implicitly as substitutes for fiducials like gold beads and quantum dots. The added value compared to previous approaches, is that the algorithm explores a huge number of random positions, which are tracked on several micrographs, while being able to identify trace failures, using a cross-validation procedure based on the 3D marker model of the tilt-series. Furthermore, this method allows the reliable identification of areas which behave as a rigid body during the tilt-series and hence addresses specific difficulties for the alignment of vitreous sections, by correcting practical caveats. The resulting alignments can attain sub-pixel precision at the local level and is able to yield a substantial number of usable tilt-series (around 60%). In principle, the algorithm has the potential to run in a fully automated fashion, and could be used to align any tilt-series directly from the microscope. Finally, we have significantly improved the user interface and implemented the source code on the graphics processing unit (GPU) to accelerate the computations.

Imaging of post-mortem human brain tissue using electron and X-ray microscopy.

Electron microscopy imaging of post-mortem human brain (PMHB) comes with a unique set of challenges due to numerous parameters beyond the researcher's control. Nevertheless, the wealth of information provided by the ultrastructural analysis of PMHB is proving crucial in our understanding of neurodegenerative diseases. This review highlights the importance of such studies and covers challenges, limitations and recent developments in the application of current EM imaging, including cryo-ET and correlative hybrid techniques, on PMHB.

TDP-43 extracted from frontotemporal lobar degeneration subject brains displays distinct aggregate assemblies and neurotoxic effects reflecting disease progression rates.

Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of people with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTLD cases into at least four subtypes, which are correlated with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for biochemical isolation of pathological TDP-43. By combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43 that become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from brain forms stable assemblies of distinct densities and morphologies that are associated with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies showed differential neurotoxicity and seeding that were correlated with disease duration of FTLD subjects. Our data are consistent with the notion that disease heterogeneity could originate from alternate pathological TDP-43 conformations, which are reminiscent of prion strains.

Reprint of "Fiducial-less alignment of cryo-sections" [J. Struct. Biol. 159 (2007) 413-423].

Cryo-electron tomography of vitreous sections is currently the most promising technique for visualizing arbitrary regions of eukaryotic cells or tissue at molecular resolution. Despite significant progress in the sample preparation techniques over the past few years, the three dimensional reconstruction using electron tomography is not as simple as in plunge frozen samples for various reasons, but mainly due to the effects of irradiation on the sections and the resulting poor alignment. Here, we present a new algorithm, which can provide a useful three-dimensional marker model after investigation of hundreds to thousands of observations calculated using local cross-correlation throughout the tilt series. The observations are chosen according to their coherence to a particular model and assigned to virtual markers. Through this type of measurement a merit figure can be calculated, precisely estimating the quality of the reconstruction. The merit figures of this alignment method are comparable to those obtained with plunge frozen samples using fiducial gold markers. An additional advantage of the algorithm is the implicit detection of areas in the sections that behave as rigid bodies and can thus be properly reconstructed.

Nanostructure of the epidermal extracellular space as observed by cryo-electron microscopy of vitreous sections of human skin.

The newly developed method, cryo-electron microscopy of vitreous sections, was used to observe the nanostructure of the epidermal extracellular space. The data were obtained from vitreous sections of freshly taken, fully hydrated, non-cryo-protected human skin. The extracellular space of viable epidermis contains desmosomes, expressing a characteristic extracellular transverse approximately 5 nm periodicity, interconnected by a relatively electron lucent inter-desmosomal space. The extracellular space between viable and cornified epidermis contains transition desmosomes at different stages of reorganization interconnected by widened areas expressing a rich variety of complex membrane-like structures. The extracellular space of cornified epidermis contains approximately 9, approximately 14, approximately 25, approximately 33, approximately 39, approximately 44, and approximately 48 nm thick regions in turn containing one, two, four, six, eight, eight, and ten parallel electron-dense lines, respectively, between adjacent corneocyte lipid envelopes. The eight-line approximately 44 nm thick regions are most prevalent.

Higher-order architecture of rhodopsin in intact photoreceptors and its implication for phototransduction kinetics.

The visual pigment rhodopsin belongs to the family of G protein-coupled receptors that can form higher oligomers. It is controversial whether rhodopsin forms oligomers and whether oligomers are functionally relevant. Here, we study rhodopsin organization in cryosections of dark-adapted mouse rod photoreceptors by cryoelectron tomography. We identify four hierarchical levels of organization. Rhodopsin forms dimers; at least ten dimers form a row. Rows form pairs (tracks) that are aligned parallel to the disk incisures. Particle-based simulation shows that the combination of tracks with fast precomplex formation, i.e. rapid association and dissociation between inactive rhodopsin and the G protein transducin, leads to kinetic trapping: rhodopsin first activates transducin from its own track, whereas recruitment of transducin from other tracks proceeds more slowly. The trap mechanism could produce uniform single-photon responses independent of rhodopsin lifetime. In general, tracks might provide a platform that coordinates the spatiotemporal interaction of signaling molecules.

Stratum corneum keratin structure, function, and formation: the cubic rod-packing and membrane templating model.

A new model for stratum corneum keratin structure, function, and formation is presented. The structural and functional part of the model, which hereafter is referred to as "the cubic rod-packing model", postulates that stratum corneum keratin intermediate filaments are arranged according to a cubic-like rod-packing symmetry with or without the presence of an intracellular lipid membrane with cubic-like symmetry enveloping each individual filament. The new model could account for (i) the cryo-electron density pattern of the native corneocyte keratin matrix, (ii) the X-ray diffraction patterns, (iii) the swelling behavior, and (iv) the mechanical properties of mammalian stratum corneum. The morphogenetic part of the model, which hereafter is referred to as "the membrane templating model", postulates the presence in cellular space of a highly dynamic small lattice parameter (<30 nm) membrane structure with cubic-like symmetry, to which keratin is associated. It further proposes that membrane templating, rather than spontaneous self-assembly, is responsible for keratin intermediate filament formation and dynamics. The new model could account for (i) the cryo-electron density patterns of the native keratinocyte cytoplasmic space, (ii) the characteristic features of the keratin network formation process, (iii) the dynamic properties of keratin intermediate filaments, (iv) the close lipid association of keratin, (v) the insolubility in non-denaturating buffers and pronounced polymorphism of keratin assembled in vitro, and (vi) the measured reduction in cell volume and hydration level between the stratum granulosum and stratum corneum. Further, using cryo-transmission electron microscopy on native, fully hydrated, vitreous epidermis we show that the subfilametous keratin electron density pattern consists, both in corneocytes and in viable keratinocytes, of one axial subfilament surrounded by an undetermined number of peripheral subfilaments forming filaments with a diameter of approximately 8 nm.

A cryotransmission electron microscopy study of skin barrier formation.

Direct visualization of the skin barrier formation process by cryotransmission electron microscopy of vitreous epidermal sections has been performed. The results obtained differ in many aspects from those of classical chemical fixation electron microscopy. Here we show that (i) a new "organelle or branched tubular structure" containing nonlamellar or partly lamellar material, and closely corresponding in numbers, dimensions, and localization to lamellar bodies of classical chemical fixation electron micrographs, and (ii) a new "ribosome complex-like structure", not preserved in classical electron micrographs, are omnipresent at apparent active sites of skin barrier formation. Evidence that skin barrier formation may not take place via extensive membrane fusion involving discrete lamellar bodies, but rather by a morphologically continuous membrane folding process are: (i) the often clearly nonlamellar content of structures corresponding to lamellar bodies with concomitant visualization of multilamellar membrane structures of the intercellular space; (ii) the "multifolded" appearance of the lipid matrix of the intercellular space; and (iii) the identification of extended "intracellular" multilamellar continuous structures with an optical density profile closely corresponding to that of the lipid matrix of the intercellular space. Based on the cryo-electron microscopic data presented in this study we propose that a membrane transition from cubic-like to multilamellar may take place already inside the "tubuloreticular cisternal membrane system" of upper granular cells recently reported by Elias et al.

Three-dimensional visualization of the molecular architecture of cell-cell junctions in situ by cryo-electron tomography of vitreous sections.

Cryo-electron tomography of vitreous sections is currently the only method for visualizing the eukaryotic ultrastructure at close to native state with molecular resolution. Here, we describe the detailed procedure of how to prepare suitable vitreous sections from mammalian skin for cryo-electron tomography, how to align the projection images of the tilt-series, and finally how to perform sub-tomogram averaging on macromolecular complexes with periodic arrangement such as desmosomes.

Structural studies on desmosomes.

Desmosomes are cadherin-based intercellular junctions that primarily provide mechanical stability to tissues such as epithelia and cardiac muscle. Desmosomal cadherins, which are Ca(2+)-dependent adhesion molecules, are of central importance in mediating direct intercellular interaction. The close association of these proteins, with intracellular components of desmosomes ultimately linked to the cytoskeleton, is believed to play an important role in tissue morphogenesis during development and wound healing. Elucidation of the binding mechanism of adhesive interfaces between the extracellular domains of cadherins has been approached by structural, biophysical and biochemical methods. X-ray crystal structures of isolated extracellular domains of cadherins have provided compelling evidence of the mutual binding of the highly conserved N-terminal residue, Trp(2), from opposing proteins. This binding interface was also implicated by biochemical and cell-adhesion assays and mutagenesis data to be the primary adhesive interface between cells. Recent results based on electron tomography of epidermal desmosomes were consistent with this view, showing cadherin molecules interacting at their N-terminal tips. An integrative structural approach involving X-ray crystallography, cryo-electron tomography and immuno-electron microscopy should give the complete picture of the architecture of this important junction; identifying its various proteins and showing their arrangements and binding interfaces under native conditions. Together with these 'static' approaches, live-cell imaging of cultured keratinocytes should provide important insights into the dynamic property of the assembly and disassembly of desmosomes.

Fiducial-less alignment of cryo-sections.

Cryo-electron tomography of vitreous sections is currently the most promising technique for visualizing arbitrary regions of eukaryotic cells or tissue at molecular resolution. Despite significant progress in the sample preparation techniques over the past few years, the three dimensional reconstruction using electron tomography is not as simple as in plunge frozen samples for various reasons, but mainly due to the effects of irradiation on the sections and the resulting poor alignment. Here, we present a new algorithm, which can provide a useful three-dimensional marker model after investigation of hundreds to thousands of observations calculated using local cross-correlation throughout the tilt series. The observations are chosen according to their coherence to a particular model and assigned to virtual markers. Through this type of measurement a merit figure can be calculated, precisely estimating the quality of the reconstruction. The merit figures of this alignment method are comparable to those obtained with plunge frozen samples using fiducial gold markers. An additional advantage of the algorithm is the implicit detection of areas in the sections that behave as rigid bodies and can thus be properly reconstructed.

Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy.

Cryo-electron microscopy of vitreous sections (CEMOVIS) has recently been shown to provide images of biological specimens with unprecedented quality and resolution. Cutting the sections remains however the major difficulty. Here, we examine the parameters influencing the quality of the sections and analyse the resulting artefacts. They are in particular: knife marks, compression, crevasses, and chatter. We propose a model taking into account the interplay between viscous flow and fracture. We confirm that crevasses are formed on only one side of the section, and define conditions by which they can be avoided. Chatter is an effect of irregular compression due to friction of the section of the knife edge and conditions to prevent this are also explored. In absence of crevasses and chatter, the bulk of the section is compressed approximately homogeneously. Within this approximation, it is possible to correct for compression by a simple linear transformation for the bulk of the section. A research program is proposed to test and refine our understanding of the sectioning process.

Cryo-electron microscopy of vitreous sections of native biological cells and tissues.

Cryo-electron microscopy of vitreous sections (CEMOVIS) is, in principle, the ultimate method of specimen preparation. It consists in ultra-rapid cooling of a sizable sample of biological material that is cut into thin sections. These are subsequently observed at low temperature in their fully hydrated vitreous state. Here, we show that CEMOVIS reveals the native state of cells and tissues with unprecedented quality and resolution. What is seen differs considerably from what conventional electron microscopy has shown previously and it is seen with more details. Our findings are demonstrated with images of cyanobacteria and skin.