[Show abstract][Hide abstract] ABSTRACT: Collective evidence argues that two members of the Nucleo-cytoplasmic large DNA viruses (NCLDVs) acquire their membrane from open membrane intermediates, postulated to be derived from membrane rupture. We now study membrane acquisition of the NCLDV African swine fever virus (ASFV). By electron tomography (ET) the virion assembles a single bilayer derived open membrane precursors that collect as ribbons in the cytoplasm. Biochemically, lumenal ER proteins are released into the cytosol, arguing that the open intermediates are ruptured ER membranes. ET shows that viral capsid assembles on the convex side of the open viral membrane to shape it into an icosahedron. The viral capsid is composed of tiny spikes with a diameter of ∼5 nm, connected to the membrane by a 6 nm wide structure displaying thin striations, as observed by several complementary EM imaging methods. Immature particles display an opening that closes after uptake of the viral genome and core proteins, followed by the formation of the mature virion. Together with our previous data this study shows a common principle of NCLDVs to build a single internal envelope from open membrane intermediates. Our data now provide biochemical evidence that these open intermediates result from rupture of a cellular membrane, the ER.
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[Show abstract][Hide abstract] ABSTRACT: TAR DNA-binding protein 43 (TDP-43) is one of the neuropathological hallmarks in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). It is present in patients' blood and cerebrospinal fluid (CSF); however, the source and clinical relevance of TDP-43 measurements in body fluids is uncertain. We investigated paired CSF and serum samples, blood lymphocytes, brain urea fractions and purified exosomes from CSF for TDP-43 by one- (1D), and two-dimensional (2D) Western immunoblotting (WB) and quantitative mass spectrometry (MRM) in patients with ALS, FTLD and non-neurodegenerative diseases. By means of 2D-WB we were able to demonstrate a similar isoform pattern of TDP-43 in lymphocytes, serum and CSF in contrast to that of brain urea fractions with TDP-43 pathology. We found that the TDP-43 CSF to blood concentration ratio is about 1:200. As a possible brain specific fraction we found TDP-43 in exosome preparations from CSF by immunoblot and MRM. We conclude that TDP-43 in CSF originates mainly from blood. Measurements of TDP-43 in CSF and blood are of minor importance as a diagnostic tool, but may be important for monitoring therapy effects of TDP-43 modifying drugs.
[Show abstract][Hide abstract] ABSTRACT: Hepcidin is the central regulator of iron homeostasis and altered hepcidin signalling results in both hereditary and acquired iron overload. While the association between iron overload and development of end-stage liver disease is well established, the underlying mechanisms are largely unknown. To improve that, we analyzed hepcidin knockout (KO) mice as a model of iron-overload associated liver disease.
Hepcidin wild type (WT) and KO mice fed with 3% carbonyl iron-containing diet starting at one month of age were compared to age-matched animals kept on standard chow. Liver histology and serum parameters were used to assess the extent of liver injury and fibrosis. Iron distribution was determined by subcellular fractionation and electron microscopy.
Among mice kept on iron-rich diet, 6 months old hepcidin KO mice (vs. WT) displayed profound hepatic iron overload (3186±411 vs. 1045±159 μg/mg tissue, p< 0.005), elevated liver enzymes (ALT: KO 128±6, WT 56±5 IU/l, p< 0.05), mild hepatic inflammation and hepatocellular apoptosis. Twelve, but not six months old KO mice fed with iron-rich diet developed moderate liver fibrosis. The liver injury was accompanied by a marked lysosomal iron overload and lysosomal fragility with release of cathepsin B into the cytoplasm. Increased p62 levels and autofluorescent iron complexes suggested impaired protein degradation. As a mechanism leading to lysosomal iron overload, the autophagy (lysosomal influx) was increased.
Hepcidin KO mice represent a novel model of iron overload-related liver diseases and implicate lysosomal injury as a crucial event in iron toxicity.
Journal of Hepatology 05/2014; DOI:10.1016/j.jhep.2014.04.034 · 10.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Naturally occurring fragments of the abundant semen proteins prostatic acid phosphatase (PAP) and semenogelins form amyloid fibrils in vitro. These fibrils boost HIV infection and may play a key role in the spread of the AIDS pandemic. However, the presence of amyloid fibrils in semen remained to be demonstrated. Here, we use state of the art confocal and electron microscopy techniques for direct imaging of amyloid fibrils in human ejaculates. We detect amyloid aggregates in all semen samples and find that they partially consist of PAP fragments, interact with HIV particles and increase viral infectivity. Our results establish semen as a body fluid that naturally contains amyloid fibrils that are exploited by HIV to promote its sexual transmission.
[Show abstract][Hide abstract] ABSTRACT: Chlamydia (C.) abortus is a widely spread pathogen among ruminants that can be transmitted to women during pregnancy leading to severe systemic infection with consecutive abortion. As a member of the Chlamydiaceae, C. abortus shares the characteristic feature of an obligate intracellular biphasic developmental cycle with two morphological forms including elementary bodies (EBs) and reticulate bodies (RBs). In contrast to other chlamydial species, C. abortus ultrastructure has not been investigated yet. To do so, samples were fixed by high-pressure freezing and processed by different electron microscopic methods. Freeze-substituted samples were analysed by transmission electron microscopy, scanning transmission electron microscopical tomography and immuno-electron microscopy, and freeze-fractured samples were analysed by cryo-scanning electron microscopy. Here, we present three ultrastructural features of C. abortus that have not been reported up to now. Firstly, the morphological evidence that C. abortus is equipped with the type three secretion system. Secondly, the accumulation and even coating of whole inclusion bodies by membrane complexes consisting of multiple closely adjacent membranes which seems to be a C. abortus specific feature. Thirdly, the formation of small vesicles in the periplasmic space of RBs in the second half of the developmental cycle. Concerning the time point of their formation and the fact that they harbour chlamydial components, these vesicles might be morphological correlates of an intermediate step during the process of redifferentiation of RBs into EBs. As this feature has also been shown for C. trachomatis and C. pneumoniae, it might be a common characteristic of the family of Chlamydiaceae.
[Show abstract][Hide abstract] ABSTRACT: In this chapter we describe three different approaches for three-dimensional imaging of electron microscopic samples: serial sectioning transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM) tomography, and focused ion beam/scanning electron microscopy (FIB/SEM) tomography. With these methods, relatively large volumes of resin-embedded biological structures can be analyzed at resolutions of a few nm within a reasonable expenditure of time. The traditional method is serial sectioning and imaging the same area in all sections. Another method is TEM tomography that involves tilting a section in the electron beam and then reconstruction of the volume by back projection of the images. When the scanning transmission (STEM) mode is used, thicker sections (up to 1 μm) can be analyzed. The third approach presented here is focused ion beam/scanning electron microscopy (FIB/SEM) tomography, in which a sample is repeatedly milled with a focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrary small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. We show that resolution of this approach is considerably improved when the secondary electron signal is used. However, the most important prerequisite for three-dimensional imaging is good specimen preparation. For all three imaging methods, cryo-fixed (high-pressure frozen) and freeze-substituted samples have been used.
[Show abstract][Hide abstract] ABSTRACT: Compared to the cytoplasmic F-actin abundance in cells, nuclear F-actin levels are generally quite low. However, nuclear actin is present in certain cell types including oocytes and under certain cellular conditions including stress or serum stimulation. Currently, the architecture and polymerization status of nuclear actin networks has not been analyzed in great detail. In this study, we investigated the architecture and functions of such nuclear actin networks. We generated nuclear actin polymers by overexpression of actin proteins fused to a nuclear localization signal (NLS). Raising nuclear abundance of a NLS wild-type actin, we observed phalloidin- and LifeAct-positive actin bundles forming a nuclear cytoskeletal network consisting of curved F-actin. In contrast, a polymer-stabilizing actin mutant (NLS-G15S-actin) deficient in interacting with the actin-binding protein cofilin generated a nuclear actin network reminiscent of straight stress fiber-like microfilaments in the cytoplasm. We provide a first electron microscopic description of such nuclear actin polymers suggesting bundling of actin filaments. Employing different cell types from various species including neurons, we show that the morphology of and potential to generate nuclear actin are conserved. Finally, we demonstrate that nuclear actin affects cell function including morphology, serum response factor-mediated gene expression, and herpes simplex virus infection. Our data suggest that actin is able to form filamentous structures inside the nucleus, which share architectural and functional similarities with the cytoplasmic F-actin.
[Show abstract][Hide abstract] ABSTRACT: Oxygen partial pressures (O2pp) inside rhizomes of Phragmites australis were measured to monitor diurnal dynamics. Root-associated biofilms were characterised regarding methane-oxidising bacteria (MOB) by immuno labelling. Runs of O2pp showed distinct diurnal patterns repeating day-to-day. Soon after sunrise O2pp increased steeply to around 185 hPa, remained on this level over mid-day and decreased exponentially over night to about 80 hPa. Root surfaces were densely packed with bacteria of which 34-37% accounted for potential MOB. This emphasises the importance of the oxic rhizosphere in the degradation of organic matter and methane oxidation and diurnal fluctuation of O2pp may implicate for variation of biogeochemical processes involved in wastewater treatment.
Desalination and water treatment 03/2013; 51(13-15):3026-3031. DOI:10.1080/19443994.2012.748452 · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intermediate filament networks are part of the cytoskeleton and protect cellular integrity during large deformations. In cells
from mesenchymal lineage the cytoskeleton is centrally involved in signal transduction, thereby influencing differentiation.
We study the ultrastructure of IF networks in three human mesenchymal cell types, namely undifferentiated mesenchymal stem
cells, chondrocytes, and osteoblasts. In order to capture the high morphological variability of IF networks we apply techniques
from image analysis to extract the network graph from 2D scanning electron microscopy (SEM) images in a fully automatic way,
which allows for a high-throughput analysis of SEM data. The extracted network graphs are analyzed by techniques from spatial
statistics to detect differences in network morphology between different cell types and infer strategies of network remodeling
used by the cells to adapt their mechanical properties during migration and differentiation.
KeywordsCytoskeleton–Intermediate filaments–Image analysis–Spatial statistics–Cell mechanics–High-throughput microscopy–Mesenchymal stem cell–Osteoblast–Chondrocyte
[Show abstract][Hide abstract] ABSTRACT: Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.
[Show abstract][Hide abstract] ABSTRACT: Using an electron microscope's scanning transmission mode (STEM) for collection of tomographic datasets is advantageous compared to bright field transmission electron microscopic (TEM). For image formation, inelastic scattering does not cause chromatic aberration, since in STEM mode no image forming lenses are used after the beam has passed the sample, in contrast to regular TEM. Therefore, thicker samples can be imaged. It has been experimentally demonstrated that STEM is superior to TEM and energy filtered TEM for tomography of samples as thick as 1 μm. Even when using the best electron microscope, adequate sample preparation is the key for interpretable results. We adapted protocols for high-pressure freezing of cultivated cells from a physiological state. In this chapter, we describe optimized high-pressure freezing and freeze substitution protocols for STEM tomography in order to obtain high membrane contrast.
[Show abstract][Hide abstract] ABSTRACT: In this paper we show how to obtain a three-dimensional model of virus-infected cells by serial sectioning of resin embedded samples and transmission electron microscopic imaging. The method bases on sample fixation by high pressure freezing and processing by freeze substitution with the goal to preserve the structures of interest close to the natural state, as previously described (Walther et al., High pressure freezing for scanning transmission electron tomography analysis of cellular organelles. In: Mossman BT, Taatjes DJ (eds) Cell imaging techniques, vol 931, Methods in molecular biology. Humana Press, Totowa, NJ, pp 525-535, 2013). Advantages of serial sectioning compared to that of other tomographic methods are as follows: No special and expensive additional equipment is required. Relatively large volumes, such as whole cells, can be three-dimensionally reconstructed in a reasonable amount of time. Serial sectioning is a non-destructive method; the sections can be stored, re-imaged, or processed for immunogold labeling when more specific data are requested or when new scientific questions are raised (e.g., higher magnifications, protein distributions). We have recently used this method to obtain a three-dimensional model of the complete assembly complex of an HCMV infected cell, which allowed a detailed insight into this virally induced compartment (Schauflinger et al., Cell Microbiol 15(2):305-314, 2013).
[Show abstract][Hide abstract] ABSTRACT: Freeze fracturing is applied to make the wetting behavior of artificially nanopatterned Si surfaces directly visible. For this purpose, hexagonally arranged nanopillars of fixed areal density (127 µm-2) and diameters (35 nm) but varying heights (40 nm to 150 nm) were fabricated on Silicon. Measurement of contact angles (CA) including hysteresis allowed to distinguish between the Wenzel-(W-) and the Cassie-Baxter (CB-) states with droplets completely wetting the pillars or just riding on top of them, respectively. It is demonstrated that freeze fracturing is able to resolve 3-dimensional features of 5 nm within the ice replica of the corresponding wetting states. In this way, laterally sharp transitions from CB- to W-states could be revealed as well as the predicted stability of W-state islands within a surrounding CB-state be experimentally confirmed on the nanoscale.