Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.

Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.

3D surface reconstruction of cellular cryo-soft X-ray microscopy tomograms using semisupervised deep learning.

Cryo-soft X-ray tomography (cryo-SXT) is a powerful method to investigate the ultrastructure of cells, offering resolution in the tens of nanometer range and strong contrast for membranous structures without requiring labeling or chemical fixation. The short acquisition time and the relatively large field of view leads to fast acquisition of large amounts of tomographic image data. Segmentation of these data into accessible features is a necessary step in gaining biologically relevant information from cryo-soft X-ray tomograms. However, manual image segmentation still requires several orders of magnitude more time than data acquisition. To address this challenge, we have here developed an end-to-end automated 3D segmentation pipeline based on semisupervised deep learning. Our approach is suitable for high-throughput analysis of large amounts of tomographic data, while being robust when faced with limited manual annotations and variations in the tomographic conditions. We validate our approach by extracting three-dimensional information on cellular ultrastructure and by quantifying nanoscopic morphological parameters of filopodia in mammalian cells.

Unveiling the Inner Structure of Micrometric Hollow Polymeric Fibers Using Synchrotron X-Ray Nanotomography.

In this study, a novel application of synchrotron X-ray nanotomography based on high-resolution full-field transmission X-ray microscopy for characterizing the structure and morphology of micrometric hollow polymeric fibers is presented. By employing postimage analysis using an open-source software such as Tomviz and ImageJ, various key parameters in fiber morphology, including diameter, wall thickness, wall thickness distribution, pore size, porosity, and surface roughness, were assessed. Electrospun polycaprolactone fibers with micrometric diameters and submicrometric features with induced porosity via gas dissolution foaming were used to this aim. The acquired synchrotron X-ray nanotomography data were analyzed using two approaches: 3D tomographic reconstruction and 2D radiographic projection-based analysis. The results of the combination of both approaches demonstrate unique capabilities of this technique, not achievable by other available techniques, allowing for a full characterization of the internal and external morphology and structure of the fibers as well as to obtain valuable qualitative insights into the overall fiber structure.

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination.

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.

Machine learning of reverse transcription signatures of variegated polymerases allows mapping and discrimination of methylated purines in limited transcriptomes.

Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.

Mode of action of quinoline antimalarial drugs in red blood cells infected by Plasmodium falciparum revealed in vivo.

The most widely used antimalarial drugs belong to the quinoline family. Their mode of action has not been characterized at the molecular level in vivo. We report the in vivo mode of action of a bromo analog of the drug chloroquine in rapidly frozen Plasmodium falciparum-infected red blood cells. The Plasmodium parasite digests hemoglobin, liberating the heme as a byproduct, toxic to the parasite. It is detoxified by crystallization into inert hemozoin within the parasitic digestive vacuole. By mapping such infected red blood cells with nondestructive X-ray microscopy, we observe that bromoquine caps hemozoin crystals. The measured crystal surface coverage is sufficient to inhibit further hemozoin crystal growth, thereby sabotaging heme detoxification. Moreover, we find that bromoquine accumulates in the digestive vacuole, reaching submillimolar concentration, 1,000-fold more than that of the drug in the culture medium. Such a dramatic increase in bromoquine concentration enhances the drug's efficiency in depriving heme from docking onto the hemozoin crystal surface. Based on direct observation of bromoquine distribution in the digestive vacuole and at its membrane surface, we deduce that the excess bromoquine forms a complex with the remaining heme deprived from crystallization. This complex is driven toward the digestive vacuole membrane, increasing the chances of membrane puncture and spillage of heme into the interior of the parasite.

Digital media in intergenerational communication: Status quo and future scenarios for the grandparent-grandchild relationship.

Communication technologies play an important role in maintaining the grandparent-grandchild (GP-GC) relationship. Based on Media Richness Theory, this study investigates the frequency of use (RQ1) and perceived quality (RQ2) of established media as well as the potential use of selected innovative media (RQ3) in GP-GC relationships with a particular focus on digital media. A cross-sectional online survey and vignette experiment were conducted in February 2021 among N = 286 university students in Germany (mean age 23 years, 57% female) who reported on the direct and mediated communication with their grandparents. In addition to face-to-face interactions, non-digital and digital established media (such as telephone, texting, video conferencing) and innovative digital media, namely augmented reality (AR)-based and social robot-based communication technologies, were covered. Face-to-face and phone communication occurred most frequently in GP-GC relationships: 85% of participants reported them taking place at least a few times per year (RQ1). Non-digital established media were associated with higher perceived communication quality than digital established media (RQ2). Innovative digital media received less favorable quality evaluations than established media. Participants expressed doubts regarding the technology competence of their grandparents, but still met innovative media with high expectations regarding improved communication quality (RQ3). Richer media, such as video conferencing or AR, do not automatically lead to better perceived communication quality, while leaner media, such as letters or text messages, can provide rich communication experiences. More research is needed to fully understand and systematically improve the utility, usability, and joy of use of different digital communication technologies employed in GP-GC relationships.

The need to freeze-Dehydration during specimen preparation for electron microscopy collapses the endothelial glycocalyx regardless of fixation method.

OBJECTIVE: The endothelial glycocalyx covers the luminal surface of the endothelium and plays key roles in vascular function. Despite its biological importance, ideal visualization techniques are lacking. The current study aimed to improve the preservation and subsequent imaging quality of the endothelial glycocalyx. METHODS: In mice, the endothelial glycocalyx was contrasted with a mixture of lanthanum and dysprosium (LaDy). Standard chemical fixation was compared with high-pressure frozen specimens processed with freeze substitution. Also, isolated brain microvessels and cultured endothelial cells were high-pressure frozen and by transmission soft x-rays, imaged under cryogenic conditions. RESULTS: The endothelial glycocalyx was in some tissues significantly more voluminous from chemically fixed specimens compared with high-pressure frozen specimens. LaDy labeling introduced excessive absorption contrast, which impeded glycocalyx measurements in isolated brain microvessels when using transmission soft x-rays. In non-contrasted vessels, the glycocalyx was not resolved. LaDy-contrasted, cultured brain endothelial cells allowed to assess glycocalyx volume in vitro. CONCLUSIONS: Both chemical and cryogenic fixation followed by dehydration lead to substantial collapse of the glycocalyx. Cryogenic fixation without freeze substitution could be a way forward although transmission soft x-ray tomography based solely on amplitude contrast seems unsuitable.

Engineering of a DNA Polymerase for Direct m6 A Sequencing.

Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N6 -methyladenosine (m6 A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m6 A-containing RNA prior to sequencing, since m6 A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m6 A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N6 -methylation. We identified a mutant that exhibits increased misincorporation opposite m6 A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m6 A sites directly from the sequencing data of untreated RNA samples.

A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure.

Recently discovered new chemical entities in RNA modifications have involved surprising functional groups that enlarge the chemical space of RNA. Using LC-MS, we found over 100 signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds that are new structures of RNA modifications. One structure was elucidated by deuterium exchange and high-resolution mass spectrometry. The structure of msms2 i6 A (2-methylthiomethylenethio-N6-isopentenyl-adenosine) was confirmed by methione-D3 feeding experiments and by synthesis of the nucleobase. The msms2 i6 A contains a thioacetal, shown in vitro to be biosynthetically derived from ms2 i6 A by the radical-SAM enzyme MiaB. This enzyme performs thiomethylation, forming ms2 i6 A from i6 A in a first turnover. The new thioacetal is formed by a second turnover. Along with the pool of 36 new modifications, this work describes a new layer of RNA modification chemistry.

Biomolecular environment, quantification, and intracellular interaction of multifunctional magnetic SERS nanoprobes.

Multifunctional composite nanoprobes consisting of iron oxide nanoparticles linked to silver and gold nanoparticles, Ag-Magnetite and Au-Magnetite, respectively, were introduced by endocytic uptake into cultured fibroblast cells. The cells containing the non-toxic nanoprobes were shown to be displaceable in an external magnetic field and can be manipulated in microfluidic channels. The distribution of the composite nanostructures that are contained in the endosomal system is discussed on the basis of surface-enhanced Raman scattering (SERS) mapping, quantitative laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping, and cryo soft X-ray tomography (cryo soft-XRT). Cryo soft-XRT of intact, vitrified cells reveals that the composite nanoprobes form intra-endosomal aggregates. The nanoprobes provide SERS signals from the biomolecular composition of their surface in the endosomal environment. The SERS data indicate the high stability of the nanoprobes and of their plasmonic properties in the harsh environment of endosomes and lysosomes. The spectra point at the molecular composition at the surface of the Ag-Magnetite and Au-Magnetite nanostructures that is very similar to that of other composite structures, but different from the composition of pure silver and gold SERS nanoprobes used for intracellular investigations. As shown by the LA-ICP-MS data, the uptake efficiency of the magnetite composites is approximately two to three times higher than that of the pure gold and silver nanoparticles.

Tomographic reconstruction in soft x-ray microscopy using focus-stack back-projection.

Tomographic reconstruction in soft x-ray microscopy is a powerful technique for obtaining high-resolution 3D images of biological samples. However, the depth of focus of such zone-plate-based microscopes is typically shorter than the thickness of many relevant biological objects, challenging the validity of the projection assumption used in conventional reconstruction algorithms. In order to make full use of the soft x-ray microscopes' high resolution, the tomographic reconstruction needs to take the depth of focus into account. Here we present a method to achieve high resolution in the full sample when the depth of focus is short compared to the sample thickness. The method relies on the back-projection of focus-stacked image data from x-ray microscopy. We demonstrate the method on theoretical and experimental data.

Influence of random zone positioning errors on the resolving power of Fresnel zone plates.

Fresnel zone plates produced by electron beam lithography and planar etching techniques provide a resolving power of about 10 nm. An alternative zone plate fabrication approach is based on alternately coating a micro-wire with two different materials. With this process, very thin zone layers with very high aspect ratios can be deposited. However, depending on the fabrication method, random zone positioning errors may introduce strong aberrations. We simulate the effect of positioning errors using different random fluctuations and study their influence on zone plate resolution. We find that random errors significantly decrease the contrast transfer of X-ray microscopes. Additionally, we derive an upper bound for the mean acceptable variance of the deposition rate.

Ergosterol promotes aggregation of natamycin in the yeast plasma membrane.

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability became compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and caused a reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogues of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. Importantly, binding and membrane aggregation of natamycin was paralleled by a decrease of the dipole potential in the PM, and this effect was enhanced in the presence of myriocin. We conclude that ergosterol promotes binding and aggregation of natamycin in the yeast PM, which can be synergistically enhanced by inhibitors of sphingolipid synthesis.

Ultrathin 2D coordination polymer nanosheets by surfactant-mediated synthesis.

Low-dimensional nanostructures offer a host of intriguing properties which are distinct from those of the bulk material, owing to size-confinement effects and amplified surface areas. Here, we report on the scalable, bottom-up synthesis of ultrathin coordination polymer nanosheets via surfactant-mediated synthesis and subsequent exfoliation. Layers of a two-dimensional (2D) zinc coordination polymer are self-assembled in the interlamellar space of a reverse microemulsion mesophase into stacks of nanosheets interleaved with cethyltrimethylammonium bromide (CTAB) at regular intervals, thus giving rise to a lamellar hybrid mesostructure with a lattice period of ~8 nm and an underlying highly crystalline substructure. The basic structural motif is composed of 2D acetato-benzimidazolato-zinc layers of tetrahedrally coordinated zinc joined together by anionic acetate and benzimidazolate ligands. The hierarchical structure was studied by PXRD, TEM, EDX, EELS, AFM, and solid-state NMR spectroscopy, revealing a high level of order on both the atomic and mesoscale, suggesting fairly strong interactions along the organic-inorganic hybrid interface. Exfoliation of the hybrid material in organic solvents such as THF and chloroform yields sheet- and belt-like nanostructures with lateral sizes between 10's and 100's of nanometers and a height of about 10 nm measured by AFM, which precisely maps the basal spacing of the lamellar mesostructure; further exfoliation results in nanobelts with minimum sizes around 4 nm. Finally, the sheetlike nanostructures behave as morphological chameleons, transforming into highly regular multiwalled coordination polymer nanotubes upon treatment with organic solvents.

Cationically charged Mn(II)Al(III) LDH nanosheets by chemical exfoliation and their use as building blocks in graphene oxide-based materials.

We report on the synthesis and exfoliation of Mn(II)Al(III) sulfonate and sulfate layered double hydroxides (LDHs) and their combination with graphene oxide by charge-directed self-assembly. The synthesis of the LDH compounds has been accomplished either directly by coprecipitation of the respective hydroxides with sulfonate anions or by ion-exchange of the chloride-containing LDH with sodium dodecylsulfate. Exfoliation of the bulk material in formamide yields colloidal suspensions of positively charged nanosheets with lateral dimensions of tens to hundreds of nanometers and thicknesses down to 1.3 nm, ascertained by TEM and AFM. Flocculation of the LDH nanosheets with an aqueous graphene oxide suspension yields a hybrid material that can be converted to a reduced graphene oxide/LDH composite by hydrazine reduction. The hybrid materials were tested for pseudocapacitive electrochemical storage capacity and electrocatalytic oxygen evolution reactions and showed significant increases compared to the pristine materials.

Kinetic modelling of sterol transport between plasma membrane and endo-lysosomes based on quantitative fluorescence and X-ray imaging data.

Niemann Pick type C1 and C2 (NPC1 and NPC2) are two sterol-binding proteins which, together, orchestrate cholesterol transport through late endosomes and lysosomes (LE/LYSs). NPC2 can facilitate sterol exchange between model membranes severalfold, but how this is connected to its function in cells is poorly understood. Using fluorescent analogs of cholesterol and quantitative fluorescence microscopy, we have recently measured the transport kinetics of sterol between plasma membrane (PM), recycling endosomes (REs) and LE/LYSs in control and NPC2 deficient fibroblasts. Here, we use kinetic modeling of this data to determine rate constants for sterol transport between intracellular compartments. Our model predicts that sterol is trapped in intraluminal vesicles (ILVs) of LE/LYSs in the absence of NPC2, causing delayed sterol export from LE/LYSs in NPC2 deficient fibroblasts. Using soft X-ray tomography, we confirm, that LE/LYSs of NPC2 deficient cells but not of control cells contain enlarged, carbon-rich intraluminal vesicular structures, supporting our model prediction of lipid accumulation in ILVs. By including sterol export via exocytosis of ILVs as exosomes and by release of vesicles-ectosomes-from the PM, we can reconcile measured sterol efflux kinetics and show that both pathways can be reciprocally regulated by the intraluminal sterol transfer activity of NPC2 inside LE/LYSs. Our results thereby connect the in vitro function of NPC2 as sterol transfer protein between membranes with its in vivo function.

Automated quantification of vacuole fusion and lipophagy in Saccharomyces cerevisiae from fluorescence and cryo-soft X-ray microscopy data using deep learning.

During starvation in the yeast Saccharomyces cerevisiae vacuolar vesicles fuse and lipid droplets (LDs) can become internalized into the vacuole in an autophagic process named lipophagy. There is a lack of tools to quantitatively assess starvation-induced vacuole fusion and lipophagy in intact cells with high resolution and throughput. Here, we combine soft X-ray tomography (SXT) with fluorescence microscopy and use a deep-learning computational approach to visualize and quantify these processes in yeast. We focus on yeast homologs of mammalian NPC1 (NPC intracellular cholesterol transporter 1; Ncr1 in yeast) and NPC2 proteins, whose dysfunction leads to Niemann Pick type C (NPC) disease in humans. We developed a convolutional neural network (CNN) model which classifies fully fused versus partially fused vacuoles based on fluorescence images of stained cells. This CNN, named Deep Yeast Fusion Network (DYFNet), revealed that cells lacking Ncr1 (ncr1∆ cells) or Npc2 (npc2∆ cells) have a reduced capacity for vacuole fusion. Using a second CNN model, we implemented a pipeline named LipoSeg to perform automated instance segmentation of LDs and vacuoles from high-resolution reconstructions of X-ray tomograms. From that, we obtained 3D renderings of LDs inside and outside of the vacuole in a fully automated manner and additionally measured droplet volume, number, and distribution. We find that ncr1∆ and npc2∆ cells could ingest LDs into vacuoles normally but showed compromised degradation of LDs and accumulation of lipid vesicles inside vacuoles. Our new method is versatile and allows for analysis of vacuole fusion, droplet size and lipophagy in intact cells.Abbreviations: BODIPY493/503: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-Indacene; BPS: bathophenanthrolinedisulfonic acid disodium salt hydrate; CNN: convolutional neural network; DHE; dehydroergosterol; npc2∆, yeast deficient in Npc2; DSC, Dice similarity coefficient; EM, electron microscopy; EVs, extracellular vesicles; FIB-SEM, focused ion beam milling-scanning electron microscopy; FM 4-64, N-(3-triethylammoniumpropyl)-4-(6-[4-{diethylamino} phenyl] hexatrienyl)-pyridinium dibromide; LDs, lipid droplets; Ncr1, yeast homolog of human NPC1 protein; ncr1∆, yeast deficient in Ncr1; NPC, Niemann Pick type C; NPC2, Niemann Pick type C homolog; OD600, optical density at 600 nm; ReLU, rectifier linear unit; PPV, positive predictive value; NPV, negative predictive value; MCC, Matthews correlation coefficient; SXT, soft X-ray tomography; UV, ultraviolet; YPD, yeast extract peptone dextrose.

Spectromicroscopy of Nanoscale Materials in the Tender X-Ray Regime Enabled by a High Efficient Multilayer-Based Grating Monochromator.

The combination of near edge X-ray absorption spectroscopy with nanoscale X-ray imaging is a powerful analytical tool for many applications in energy technologies, catalysis, which are critical to combat climate change, as well as microelectronics and life science. Materials from these scientific areas often contain key elements, such as Si, P, S, Y, Zr, Nb, and Mo as well as lanthanides, whose X-ray absorption edges lie in the so-called tender photon energy range 1.5-5.0 keV. Neither conventional grazing incidence grating nor crystal monochromators have high transmission in this energy range, thereby yielding the tender photon energy gap. To close this gap, a monochromator setup based on a multilayer coated blazed plane grating and plane mirror is devised. The measurements show that this novel concept improves the photon flux in the tender X-ray regime by two-orders-of-magnitude enabling previously unattainable laboratory and synchrotron-based studies. This setup is applied to perform nanoscale spectromicroscopy studies. The high photon flux provides sufficient sensitivity to obtain the electronic structure of Mo in platinum-free MoNi4 nanoparticles for electrochemical energy conversion. Additionally, it is shown that the chemical bonding of nano-structures in integrated circuits can be distinguished by the electronic configuration at the Si-K edge.