UVC inactivation of pathogenic samples suitable for cryo-EM analysis.

Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the ultrastructure of the sample.

Optimized cryo-EM data-acquisition workflow by sample-thickness determination.

Sample thickness is a known key parameter in cryo-electron microscopy (cryo-EM) and can affect the amount of high-resolution information retained in the image. Yet, common data-acquisition approaches in single-particle cryo-EM do not take it into account. Here, it is demonstrated how the sample thickness can be determined before data acquisition, allowing the identification of optimal regions and the restriction of automated data collection to images with preserved high-resolution details.

Cell death induced autophagy contributes to terminal differentiation of skin and skin appendages.

In the adult mammalian skin, cells are constantly renewing, differentiating and moving upward, to finally die in a yet not fully understood manner. Here, we provide evidence that macroautophagy/autophagy has a dual role in the skin. In addition to its known catabolic protective role as an evolutionary conserved upstream regulator of lysosomal degradation, we show that autophagy induced cell death (CDA) occurs in epithelial lineage-derived organs, such as the inter-follicular epidermis, the sebaceous- and the Harderian gland.

Software for automated acquisition of electron tomography tilt series.

For automated acquisition of tilt series for electron tomography, software needs to handle complications such as movements of the sample in x/y and z, increased projected thickness at high tilt, specimen drift, etc. In addition, many applications require special functionality such as low dose acquisition, automated sequential (batch) tomography, or montage tomography. After reviewing how these difficulties can be addressed and a closer look at what advanced acquisition strategies are employed in biosciences, this chapter introduces acquisition software both developed in academia as well as by hardware vendors.

Subcellular analysis of pigeon hair cells implicates vesicular trafficking in cuticulosome formation and maintenance.

Hair cells are specialized sensors located in the inner ear that enable the transduction of sound, motion, and gravity into neuronal impulses. In birds some hair cells contain an iron-rich organelle, the cuticulosome, that has been implicated in the magnetic sense. Here, we exploit histological, transcriptomic, and tomographic methods to investigate the development of cuticulosomes, as well as the molecular and subcellular architecture of cuticulosome positive hair cells.

No evidence for intracellular magnetite in putative vertebrate magnetoreceptors identified by magnetic screening.

The cellular basis of the magnetic sense remains an unsolved scientific mystery. One theory that aims to explain how animals detect the magnetic field is the magnetite hypothesis. It argues that intracellular crystals of the iron oxide magnetite (Fe3O4) are coupled to mechanosensitive channels that elicit neuronal activity in specialized sensory cells.

Autophagy facilitates secretion and protects against degeneration of the Harderian gland.

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice.

Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial.

Mechanosensing through focal adhesion-anchored intermediate filaments.

Integrin-based mechanotransduction involves a complex focal adhesion (FA)-associated machinery that is able to detect and respond to forces exerted either through components of the extracellular matrix or the intracellular contractile actomyosin network. Here, we show a hitherto unrecognized regulatory role of vimentin intermediate filaments (IFs) in this process. By studying fibroblasts in which vimentin IFs were decoupled from FAs, either because of vimentin deficiency (V0) or loss of vimentin network anchorage due to deficiency in the cytolinker protein plectin (P0), we demonstrate attenuated activation of the major mechanosensor molecule FAK and its downstream targets Src, ERK1/2, and p38, as well as an up-regulation of the compensatory feedback loop acting on RhoA and myosin light chain.

Nanocarriers for dermal drug delivery: influence of preparation method, carrier type and rheological properties.

Nanocarriers are highly interesting delivery systems for the dermal application of drugs. Based on a eudermic alkylpolyglycosid nanoemulsions, solid lipid nanoparticles (SLN) and nano-structured lipid carriers (NLC) were prepared by ultrasonic dispersion. The ultrasound preparation technique turned out to be convenient and rapid.

Actin branching in the initiation and maintenance of lamellipodia.

Using correlated live-cell imaging and electron tomography we found that actin branch junctions in protruding and treadmilling lamellipodia are not concentrated at the front as previously supposed, but link actin filament subsets in which there is a continuum of distances from a junction to the filament plus ends, for up to at least 1 μm. When branch sites were observed closely spaced on the same filament their separation was commonly a multiple of the actin helical repeat of 36 nm. Image averaging of branch junctions in the tomograms yielded a model for the in vivo branch at 2.

Optimisation of multiple W/O/W nanoemulsions for dermal delivery of aciclovir.

In the present study multiple W/O/W nanoemulsions were optimised for the dermal application of the antiviral drug aciclovir. The phase inversion temperature method was employed to prepare the formulations without the input of high pressure. During formulation design the ethoxylated surfactants were varied and if possible partly replaced by natural sugar surfactants.

The transcription factor c-Jun protects against sustained hepatic endoplasmic reticulum stress thereby promoting hepatocyte survival.

Unlabelled: Endoplasmic reticulum (ER) stress due to accumulation of hepatoviral or misfolded proteins is increasingly recognized as an important step in the pathogenesis of inflammatory, toxic, and metabolic liver diseases. ER stress results in the activation of several intracellular signaling pathways including Jun N-terminal kinase (JNK). The AP-1 (activating protein 1) transcription factor c-Jun is a prototypic JNK target and important regulator of hepatocyte survival, proliferation, and liver tumorigenesis.

Modification of the conformational skin structure by treatment with liposomal formulations and its correlation to the penetration depth of aciclovir.

The stratum corneum (SC), top layer of the epidermis, is comprised mostly of lipids that are responsible for the permeability properties of the SC and which protect the body from external agents. Changes in these skin microconstituents can be understood by instrumental methods such as attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy. The present work shows that different types of analyzed skin, dermatomed abdominal porcine skin, pig ear skin, and human heat separated skin, influenced both the shape and the intensity of recorded spectra.

The actin cytoskeleton in whole mount preparations and sections.

In non-muscle cells, the actin cytoskeleton plays a key role by providing a scaffold contributing to the definition of cell shape, force for driving cell motility, cytokinesis, endocytosis, and propulsion of pathogens, as well as tracks for intracellular transport. A thorough understanding of these processes requires insight into the spatial and temporal organisation of actin filaments into diverse higher-order structures, such as networks, parallel bundles, and contractile arrays. Transmission and scanning electron microscopy can be used to visualise the actin cytoskeleton, but due to the delicate nature of actin filaments, they are easily affected by standard preparation protocols, yielding variable degrees of ultrastructural preservation.

Electron tomography reveals unbranched networks of actin filaments in lamellipodia.

Eukaryotic cells can initiate movement using the forces exerted by polymerizing actin filaments to extend lamellipodial and filopodial protrusions. In the current model, actin filaments in lamellipodia are organized in a branched, dendritic network. We applied electron tomography to vitreously frozen 'live' cells, fixed cells and cytoskeletons, embedded in vitreous ice or in deep-negative stain.

Automated monitoring to reduce electron microscope downtime.

High-end transmission electron microscopes are complex and sensitive instruments. Failure of one of the external supplies, malfunction of the microscope hardware or maloperation are typical reasons for subsystems to fail. Especially if undiscovered for a longer period of time, this can cause unnecessary downtime, compromising user access and increasing operating costs.

Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation.

Vasodilator-stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration-dependent manner but was inhibited by low concentrations of capping protein (CP).

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes.

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly.

The comings and goings of actin: coupling protrusion and retraction in cell motility.

Cells utilize actin filaments to produce protrusive and contractile arrays that cooperate to drive cell motility. The generation of the two arrays and the coupling between them result from the unique properties of the lamellipodium, a protrusive leaflet of cytoplasm at the cell edge. From the lamellipodium into the lamella behind, there is a transition from a fast retrograde flow of actin polymer driven by polymerization to a slow flow driven by the interaction of anti-parallel arrays of actin with myosin.