Supersensitive Multifluorophore RNA-FISH for Early Virus Detection and Flow-FISH by Using Click Chemistry.

The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid-19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single-fluorophore-containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off-target binding effects that create background noise. Here, we used click chemistry and alkyne-modified DNA oligonucleotides to prepare multiple-fluorophore-containing probes. We found that these multiple-dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5-10) of probe strands. The new method enabled the in situ detection of viral transcripts as early as 4 hours after infection.

A Photoswitchable Trivalent Cluster Mannoside to Probe the Effects of Ligand Orientation in Bacterial Adhesion.

We have recently demonstrated, by employing azobenzene glycosides, that bacterial adhesion to surfaces can be switched through reversible reorientation of the carbohydrate ligands. To investigate this phenomenon further, we have turned here to more complex-that is, multivalent-azobenzene glycoclusters. We report on the synthesis of a photosensitive trivalent cluster mannoside conjugated to an azobenzene hinge at the focal point. Molecular dynamics studies suggested that this cluster mannoside, despite the conformational flexibility of the azobenzene-glycocluster linkage, offers the potential for reversibly changing the glycocluster's orientation on a surface. Next, the photoswitchable glycocluster was attached to human cells, and adhesion assays with type 1 fimbriated Escherichia coli bacteria were performed. They showed marked differences in bacterial adhesion, dependent on the light-induced reorientation of the glycocluster moiety. These results further underline the importance of orientational effects in carbohydrate recognition and likewise the value of photoswitchable glycoconjugates for their study.

Quantification of volume and lipid filling of intracellular vesicles carrying the ABCA3 transporter.

The ABCA3 lipid transporter is located in the limiting membrane of lamellar bodies (LBs) in type-II-pneumocytes. Mutations within the ABCA3 gene may functionally impair the transporter, causing lung diseases in newborns, children and adults. Assays to quantify volume and lipid filling of the LBs on the level of the vesicular structures and thereby assess the function of ABCA3 are still lacking. In the present study human influenza haemagglutinin- (HA-) tagged wild type and mutant ABCA3 proteins were stably expressed in lung A549 cells. Fluorescently-labelled TopFluor phosphatidylcholine (TopF-PC) incorporated in surfactant-like liposomes was delivered to the cells and visualized by confocal microscopy. Subsequently, a comprehensive image analysis method was applied to quantify volume and fluorescence intensity of TopF-PC in ABCA3-HA-positive vesicles. TopF-PC accumulated within the vesicles in a time and concentration-dependent manner, whereas the volume remained unchanged, suggesting active transport into preformed ABCA3 containing vesicles. Furthermore, this finding was supported by a decrease of the fluorescence intensity within the vesicles when either the ATPase of the transporter was inhibited by vanadate, or when a disease-causing mutation (K1388N) close to the ABCA3-nucleotide binding domain 2 was introduced. Conversely, a mutation (E292V) located in the first cytoplasmic loop of ABCA3 did not significantly affect lipid transport, but rather resulted in smaller vesicles. In addition to these findings, the assay used in this work for analysing the PC-lipid transport into ABCA3 positive vesicles will be useful to screen for compounds susceptible to restore function in mutated ABCA3 protein.

En route from artificial to natural: Evaluation of inhibitors of mannose-specific adhesion of E. coli under flow.

We investigated the properties of six Escherichia coli adhesion inhibitors under static and under flow conditions. On mannan-covered model substrates and under static conditions, all inhibitors were able to almost completely abolish lectin-mediated E. coli adhesion. On a monolayer of living human microvascular endothelial cells (HMEC-1), the inhibitors reduced adhesion under static conditions as well, but a large fraction of bacteria still managed to adhere even at highest inhibitor concentrations. In contrast, under flow conditions E. coli did not exhibit any adhesion to HMEC-1 not even at inhibitor concentrations where significant adhesion was detected under static conditions. This indicates that the presence of shear stress strongly affects inhibitor properties and must be taken into account when evaluating the potency of bacterial adhesion inhibitors.

Dendrimer-Based Signal Amplification of Click-Labelled DNA in Situ.

The in vivo incorporation of alkyne-modified bases into the genome of cells is today the basis for the efficient detection of cell proliferation. Cells are grown in the presence of ethinyl-dU (EdU), fixed and permeabilised. The incorporated alkynes are then efficiently detected by using azide-containing fluorophores and the CuI -catalysed alkyne-azide click reaction. For a world in which constant improvement in the sensitivity of a given method is driving diagnostic advancement, we developed azide- and alkyne-modified dendrimers that allow the establishment of sandwich-type detection assays that show significantly improved signal intensities and signal-to-noise ratios far beyond that which is currently possible.

Azido Pentoses: A New Tool To Efficiently Label Mycobacterium tuberculosis Clinical Isolates.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (Tb), has a complex cell envelope which forms an efficient barrier to antibiotics, thus contributing to the challenges of anti-tuberculosis therapy. However, the unique Mtb cell wall can be considered an advantage and be utilized to selectively label Mtb bacteria. Here we introduce three azido pentoses as new compounds for metabolic labeling of Mtb: 3-azido arabinose (3AraAz), 3-azido ribose (3RiboAz), and 5-azido arabinofuranose (5AraAz). 5AraAz demonstrated the highest level of Mtb labeling and was efficiently incorporated into the Mtb cell wall. All three azido pentoses can be easily used to label a variety of Mtb clinical isolates without influencing Mtb-dependent phagosomal maturation arrest in infection studies with human macrophages. Thus, this metabolic labeling method offers the opportunity to attach desired molecules to the surface of Mtb bacteria in order to facilitate investigation of the varying virulence characteristics of different Mtb clinical isolates, which influence the outcome of a Tb infection.

Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sites.

The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley's L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.

Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single-Molecule Tracking Study.

We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper-free click chemistry. Finally, these biotin-tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin-induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA-protein networks employing single-molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface.

Microdomain Formation Controls Spatiotemporal Dynamics of Cell-Surface Glycoproteins.

The effect of galectin-mediated microdomain formation on the spatiotemporal dynamics of glycosylated membrane proteins in human microvascular endothelial cells (HMEC-1) was studied qualitatively and quantitatively by high-resolution fluorescence microscopy and artificially mimicked by metabolic glycoprotein engineering. Two types of membrane proteins, sialic acid-bearing proteins (SABPs) and mucin-type proteins (MTPs), were investigated. For visualization they were metabolically labeled with azido sugars and then coupled to a cyclooctyne-conjugated fluorescent dye by click chemistry. Both spatial (diffusion) and temporal (residence time) dynamics of SABPs and MTPs on the membrane were investigated after treatment with exogenous galectin-1 or -3. Strong effects of galectin-mediated lattice formation were observed for MTPs (decreased spatial mobility), but not for SABPs. Lattice formation also strongly decreased the turnover of MTPs (increased residence time on the cell membrane). The effects of galectin-mediated crosslinking was accurately mimicked by streptavidin-mediated crosslinking of biotin-tagged glycoproteins and verified by single-molecule tracking. This technique allows the induction of crosslinking of membrane proteins under precisely controlled conditions, thereby influencing membrane residence time and the spatial dynamics of glycans on the cell membrane in a controlled way.

Cell-penetrating and neurotargeting dendritic siRNA nanostructures.

We report the development of dendritic siRNA nanostructures that are able to penetrate even difficult to transfect cells such as neurons with the help of a special receptor ligand. The nanoparticles elicit strong siRNA responses, despite the dendritic structure. An siRNA dendrimer directed against the crucial rabies virus (RABV) nucleoprotein (N protein) and phosphoprotein (P protein) allowed the suppression of the virus titer in neurons below the detection limit. The cell-penetrating siRNA dendrimers, which were assembled using click chemistry, open up new avenues toward finding novel molecules able to cure this deadly disease.

Fine-tuning of proton sponges by precise diaminoethanes and histidines in pDNA polyplexes.

The cationizable nature of 'proton-sponge' transfection agents facilitates pDNA delivery in several steps. Protonated amines account for electrostatic DNA binding and cellular uptake, buffering amines mediate polyplex escape from acidifying intracellular vesicles. As demonstrated with a sequence-defined library of oligo(ethanamino)amides containing selected oligoethanamino acids and histidines, the total protonation capacity as well as the cationization pH profile within the endolysosomal range have critical impact on gene transfer. Building blocks with even numbered amine groups (Gtt, Sph) exhibited higher total endolysosomal buffer capacity than odd number (Stp) analogs. Within the endolysosomal range, Gtt has the highest buffer capacity around pH5, whereas Stp has its maximum around pH7. Histidines increased the total buffer capacity, resulted in a more continuous cationization pH profile and greatly improved transgene expression in vitro and in vivo. Using receptor targeted and polyethylene glycol shielded polyplexes, better endosomal escape and >100-fold enhanced transfection was detected. FROM THE CLINICAL EDITOR: Proton-sponge transfection agents for pDNA delivery are characterized in this study, demonstrating over 100-fold enhanced transection and better endosomal escape by using receptor targeted and polyethylene glycol shielded polyplexes.

Targeted drug delivery in cancer cells with red-light photoactivated mesoporous silica nanoparticles.

Mesoporous nanoparticles for drug delivery would benefit significantly from further improvements in targeting efficiency and endosomal release. We present a system based on colloidal mesoporous silica nanoparticles with targeting-ligands and a red-light photosensitizer. This nanoparticle system provides spatial and temporal control of the release of drugs into the cytosol of cancer cells. Furthermore, the system presents a general platform since it can be loaded with different cargos and adapted for targeting of multiple cell types.

Super-resolved fluorescence microscopy: Nobel Prize in Chemistry 2014 for Eric Betzig, Stefan Hell, and William†E. Moerner.

A big honor for small objects: The Nobel Prize in Chemistry 2014 was jointly awarded to Eric Betzig, Stefan Hell, and William E. Moerner "for the development of super-resolved fluorescence microscopy". This Highlight describes how the field of super-resolution microscopy developed from the first detection of a single molecule in 1989 to the sophisticated techniques of today.

Uptake kinetics and nanotoxicity of silica nanoparticles are cell type dependent.

In this study, it is shown that the cytotoxic response of cells as well as the uptake kinetics of nanoparticles (NPs) is cell type dependent. We use silica NPs with a diameter of 310 nm labeled with perylene dye and 304 nm unlabeled particles to evaluate cell type-dependent uptake and cytotoxicity on human vascular endothelial cells (HUVEC) and cancer cells derived from the cervix carcinoma (HeLa). Besides their size, the particles are characterized concerning homogeneity of the labeling and their zeta potential. The cellular uptake of the labeled NPs is quantified by imaging the cells via confocal microscopy in a time-dependent manner, with subsequent image analysis via a custom-made and freely available digital method, Particle_in_Cell-3D. We find that within the first 4 h of interaction, the uptake of silica NPs into the cytoplasm is up to 10 times more efficient in HUVEC than in HeLa cells. Interestingly, after 10 or 24 h of interaction, the number of intracellular particles for HeLa cells by far surpasses the one for HUVEC. Inhibitor studies show that these endothelial cells internalize 310 nm SiO2 NPs via the clathrin-dependent pathway. Remarkably, the differences in the amount of taken up NPs are not directly reflected by the metabolic activity and membrane integrity of the individual cell types. Interaction with NPs leads to a concentration-dependent decrease in mitochondrial activity and an increase in membrane leakage for HUVEC, whereas HeLa cells show only a reduced mitochondrial activity and no membrane leakage. In addition, silica NPs lead to HUVEC cell death while HeLa cells survive. These findings indicate that HUVEC are more sensitive than HeLa cells upon silica NP exposure.

Visualizing single-molecule diffusion in mesoporous materials.

Periodic mesoporous materials formed through the cooperative self-assembly of surfactants and framework building blocks can assume a variety of structures, and their widely tuneable properties make them attractive hosts for numerous applications. Because the molecular movement in the pore system is the most important and defining characteristic of porous materials, it is of interest to learn about this behaviour as a function of local structure. Generally, individual fluorescent dye molecules can be used as molecular beacons with which to explore the structure of--and the dynamics within--these porous hosts, and single-molecule fluorescence techniques provide detailed insights into the dynamics of various processes, ranging from biology to heterogeneous catalysis. However, optical microscopy methods cannot directly image the mesoporous structure of the host system accommodating the diffusing molecules, whereas transmission electron microscopy provides detailed images of the porous structure, but no dynamic information. It has therefore not been possible to 'see' how molecules diffuse in a real nanoscale pore structure. Here we present a combination of electron microscopic mapping and optical single-molecule tracking experiments to reveal how a single luminescent dye molecule travels through linear or strongly curved sections of a mesoporous channel system. In our approach we directly correlate porous structures detected by transmission electron microscopy with the diffusion dynamics of single molecules detected by optical microscopy. This opens up new ways of understanding the interactions of host and guest.

Direct visualization of dye and oligonucleotide diffusion in silica filaments with collinear mesopores.

The diffusion dynamics of terrylene diimide (TDI) dye molecules and dye-labeled double-strand DNA were studied in micrometer long silica filaments containing collinear, oriented mesopores using single molecule fluorescence microscopy. TDI was used as a stable and hydrophobic probe molecule for single molecule structural analysis. We used template-free mesoporous silica filaments with 4 nm pore diameter and chemical functionalization with one or two types of trialkoxysilane groups to enhance the affinity between the host system and the guest molecules. Insights about the mesoporous structure as well as the translational and orientational diffusion dynamics of the guest molecules observed along micrometer long trajectories could be obtained. Additionally, the stability of DNA oligomers (15 base pairs, bp, about 5.3 nm long) within the mesopores was examined, showing no degradation of the oligonucleotide upon incorporation into the mesopores. Diffusion of both guest molecules could be controlled by exposure to vapors of water or chloroform; the latter both induced a reversible on-off control of the translational movement of the molecules.

Tuning nanoparticle uptake: live-cell imaging reveals two distinct endocytosis mechanisms mediated by natural and artificial EGFR targeting ligand.

Therapeutic nanoparticles can be directed to cancer cells by incorporating selective targeting ligands. Here, we investigate the epidermal growth factor receptor (EGFR)-mediated endocytosis of gene carriers (polyplexes) either targeted with natural EGF or GE11, a short synthetic EGFR-binding peptide. Highly sensitive live-cell fluorescence microcopy with single particle resolution unraveled the existence of two different uptake mechanisms; EGF triggers accelerated nanoparticle endocytosis due to its dual active role in receptor binding and signaling activation. For GE11, an alternative EGFR signaling independent, actin-driven pathway is presented.

Enhanced bioactivity of internally functionalized cationic dendrimers with PEG cores.

Hybrid dendritic-linear block copolymers based on a 4-arm poly(ethylene glycol) (PEG) core were synthesized using an accelerated AB2/CD2 dendritic growth approach through orthogonal amine/epoxy and thiol-yne chemistries. The biological activity of these 4-arm and the corresponding 2-arm hybrid dendrimers revealed an enhanced, dendritic effect with an exponential increase in cell internalization concomitant with increasing amine end groups and low cytotoxicity. Furthermore, the ability of these hybrid dendrimers to induce endosomal escape combined with their facile and efficient synthesis makes them attractive platforms for gene transfection. The 4-arm-based dendrimer showed significantly improved DNA binding and gene transfection capabilities in comparison with the 2-arm derivative. These results combined with the MD simulation indicate a significant effect of both the topology of the PEG core and the multivalency of these hybrid macromolecules on their DNA binding and delivery capablities.

Single-particle tracking as a quantitative microscopy-based approach to unravel cell entry mechanisms of viruses and pharmaceutical nanoparticles.

Highly sensitive fluorescence microscopy techniques allow single nanoparticles to be tracked during their uptake into living cells with high temporal and spatial resolution. From analysis of the trajectories, random motion can be discriminated from active transport and the average transport velocity and/or diffusion coefficient determined. Such an analysis provides important information regarding the uptake pathway and location of viruses and nanoparticles. In this review, we give an introduction into single-particle tracking (SPT) and determination of the mean-squared displacement. We also give an overview of recent advances in SPT. These include millisecond alternating-laser excitation for removal of spectral crosstalk, alternating wide-field (WF), and total internal reflection fluorescence (TIRF) microscopy for sensitive experiments at the plasma membrane and three-dimensional tracking strategies. Throughout the review, we highlight recent advances regarding the entry (and egress) of natural and artificial viruses obtained via SPT.

Dynamics of HIV-1 assembly and release.

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1-2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of approximately 5 x 10(-3) s(-1), corresponding to 8-9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at approximately 1,500+/-700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.