[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: 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. · 0.99 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: 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: 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: 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.
[Show abstract][Hide abstract] ABSTRACT: Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium (M.) avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay, and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.
[Show abstract][Hide abstract] ABSTRACT: Electron microscopy (EM) allows visualization of viruses in fixed cells with high resolution. High pressure freezing for sample fixation in combination with freeze substitution and embedding in resin improves significantly the preservation of cellular structures and specifically of membranes. This advancement allows better visualization of human cytomegalovirus (HCMV) morphogenesis occurring at membranes. To obtain comprehensive information on viral phenotypes from ultrastructural images it is important to also quantify morphological phenotypes. This again can be much refined by three-dimensional visualization after serial sectioning. For elucidation of dynamic processes three-dimensional tomography is extremely helpful. We analyzed interaction of HCMV particles with host cell membranes during final envelopment. Both, wild-type virus and a viral mutant with impaired envelopment were analyzed in fibroblasts, but also using in vivo relevant human endothelial cells and macrophages. The quantification of the EM data showed similar ultrastructural phenotypes regarding the envelopment efficiency in the different cell types indicating similar mechanisms in late stages of virus morphogenesis. Furthermore, thorough analysis of the viral assembly complex (AC) - a virus induced cytosolic structure - by using three-dimensional visualization techniques combined with a quantitative analysis revealed that the events of final envelopment are equally distributed within the AC irrespective of different local membrane composition.
[Show abstract][Hide abstract] ABSTRACT: All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.
[Show abstract][Hide abstract] ABSTRACT: Wetland plants create partly aerobic conditions in the rhizosphere by releasing oxygen to the waterlogged substrate. The present study was conducted to characterise the arrangement of rhizobacteria, especially those active in methane oxidation, in root-associated biofilms of wetland plants. Root cross sections sampled from Typha latifolia L. (broadleafed cattail) and Phragmites australis (Cav.) Trin. ex Steud. (common reed) were scanned using light and electron microscopy. Methane-oxidising bacteria were identified and quantified by immunological labelling of the α-subunit of the methanol dehydrogenase (α-MDH; encoded in mxaF). On roots of both species there was a diverse subset of bacteria arranged in a microbial biofilm around the roots’ exodermis. Similar bacterial densities in the root-associated biofilm were detected in more basal regions and closer to the root tip. Many microbes carried notable internal membrane systems that are characteristic of methanotrophic bacteria. This morpho-anatomical characterisation was confirmed by immunogold labelling with α-MDH antibodies. Quantification of labelled bacteria revealed that 34–43% of the biofilm bacteria were potentially capable of methane turnover. These findings confirm the presence of methane-oxidising bacteria in the root-associated biofilms of the two common macrophytes T. latifolia and P. australis. This implies that the methanotrophs participate essentially in the microbial processes related to oxygen-releasing roots of wetland plants.
Flora - Morphology Distribution Functional Ecology of Plants 11/2012; 207(11):775–782. · 1.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophages (M) are first targets during human cytomegalovirus (HCMV) infection and are thought to be crucial for viral persistence and dissemination. However, since M are also a first line of defense and key modulators of the immune response, these cells are at the crossroad between protection and viral pathogenesis. To date, the M-specific contribution to the immune response against HCMV is still poorly understood. In view of the opposite roles of M1- and M2-M during initiation and resolution of the immune response, we characterized the effects of HCMV infection on classically activated M1-M and alternatively activated M2-M. Although HCMV susceptibility was higher in M2-M, HCMV established a productive and persistent infection in both types of M. Upon HCMV encounter, both types of M acquired similar features of classical activation and secreted high levels of pro-inflammatory cytokines and chemokines. As a functional consequence, conditioned media obtained from HCMV-infected M1- and M2-M potently activated freshly isolated monocytes. Finally, compared to HCMV-infected monocyte-derived dendritic cells, infected M1- and M2-M were more efficient in stimulating proliferation of autologous T cells from HCMV-seropositive donors at early times (24 hours) post infection, while the M immunostimulatory properties were reduced, but not abrogated, at later times (72 hours post infection). In summary, our findings indicate that M preserve proper antigen presentation capacity upon HCMV infection while enhancing inflammation, thus suggesting that M play a role in the maintenance of the large HCMV-specific T-cell repertoire in seropositive individuals.
[Show abstract][Hide abstract] ABSTRACT: Dominant negative (DN) mutants are powerful tools for studying essential protein-protein interactions. A systematic genetic screen of the essential murine cytomegalovirus (MCMV) protein, pM53, identified the accumulation of inhibitory mutations within conserved regions (CR) 2 and 4. The strong inhibitory potential of these CR4 mutants is characterized by a particular phenotype. The DN effect of the small insertion mutations in CR2 was too weak to analyze (M. Popa, Z. Ruzsics, M. Lötzerich, L. Dölken, C. Buser, P. Walther, and U. H. Koszinowski, J. Virol. 84:9035-9046, 2010); therefore, the present study describes the construction of M53 alleles lacking CR2 (either completely or partially) and subsequent examination of the DN effect on MCMV replication upon conditional expression. Overexpression of CR2-deficient pM53 inhibited virus production by about 10,000-fold. This was due to interference with capsid export from the nucleus and viral genome cleavage/packaging. In addition, the fate of the nuclear envelopment complex in the presence of DN pM53 overexpression was analyzed. The CR2 mutants were able to bind to pM50, albeit to a lesser extent than the wild-type protein, and re-localized the wild-type nuclear envelope complex in infected cells. Unlike the CR4 DN, the CR2 DN mutants did not affect the stability of pM50.
[Show abstract][Hide abstract] ABSTRACT: Parapodia of the sacoglossan slug Elysia timida were preserved by high-pressure cryofixation during feeding experiments and investigated with transmission electron microscopy. This slug has been known for its long-term retention of active chloroplasts and photosynthesis. We observed different stages of phagocytosis of chloroplast components from ingested algal food by slug digestive gland cells. Thylakoid stacks and stroma of chloroplasts were engulfed by the slug cells. In the slug cells thylakoids were surrounded by one membrane only. This membrane is interpreted as having been generated by the mollusk during phagocytosis. It is inferred to be eukaryotic in origin and unlikely, therefore, to be endowed with the translocons system ordinarily regulating import of algal gene-encoded plastid preproteins. Our structural findings suggest that chloroplast components in the slug cells are thylakoid stacks with chloroplast stroma only.
[Show abstract][Hide abstract] ABSTRACT: Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the 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 arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.
[Show abstract][Hide abstract] ABSTRACT: Hydrocephalus formation is a frequent complication of neuropathological insults associated with neuroinflammation. However, the mechanistic role of neuroinflammation in hydrocephalus development is unclear. We have investigated the function of the proinflammatory acting inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling system in neuroinflammatory processes and generated a novel mouse model that allows conditional activation of the IKK/NF-κB system in astrocytes. Remarkably, NF-κB activation in astrocytes during early postnatal life results in hydrocephalus formation and additional defects in brain development. NF-κB activation causes global neuroinflammation characterized by a strong, astrocyte-specific expression of proinflammatory NF-κB target genes as well as a massive infiltration and activation of macrophages. In this animal model, hydrocephalus formation is specifically induced during a critical time period of early postnatal development, in which IKK/NF-κB-induced neuroinflammation interferes with ependymal ciliogenesis. Our findings demonstrate for the first time that IKK/NF-κB activation is sufficient to induce hydrocephalus formation and provides a potential mechanistic explanation for the frequent association of neuroinflammation and hydrocephalus formation during brain development, namely impairment of ependymal cilia formation. Therefore, our study might open up new perspectives for the treatment of certain types of neonatal and childhood hydrocephalus associated with hemorrhages and infections.
Journal of Neuroscience 08/2012; 32(34):11511-23. · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanical properties of epithelial cells are modulated by structural changes in keratin intermediate filament networks. To investigate the relationship between network architecture and viscoelasticity, we assembled keratin filaments from recombinant keratin proteins 8 (K8) and 18 (K18) in the presence of divalent ions (Mg(2+)). We probed the viscoelastic modulus of the network by tracking the movement of microspheres embedded in the network during assembly, and studied the network architecture using scanning electron microscopy. Addition of Mg(2+) at physiological concentrations (<1 mM) resulted in networks whose structure was similar to that of keratin networks in epithelial cells. Moreover, the elastic moduli of networks assembled in vitro were found to be within the same magnitude as those measured in keratin networks of detergent-extracted epithelial cells. These findings suggest that Mg(2+)-induced filament cross-linking represents a valid model for studying the cytoskeletal mechanics of keratin networks.