Super-resolution microscopy reveals specific recruitment of HIV-1 envelope proteins to viral assembly sites dependent on the envelope C-terminal tail

The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread . Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse

Computational Functional Genomics-Based AmpliSeq™ Panel for Next-Generation Sequencing of Key Genes of Pain

The genetic background of pain is becoming increasingly well understood, which opens up possibilities for predicting the individual risk of persistent pain and the use of tailored therapies adapted to the variant pattern of the patient’s pain-relevant genes. The individual variant pattern of pain-relevant genes is accessible via next-generation sequencing, although the analysis of all “pain genes” would be expensive. Here, we report on the development of a cost-effective next generation sequencing-based pain-genotyping assay comprising the development of a customized AmpliSeq™ panel and bioinformatics approaches that condensate the genetic information of pain by identifying the most representative genes. The panel includes 29 key genes that have been shown to cover 70% of the biological functions exerted by a list of 540 so-called “pain genes” derived from transgenic mice experiments. These were supplemented by 43 additional genes that had been independently proposed as relevant for persistent pain. The functional genomics covered by the resulting 72 genes is particularly represented by mitogen-activated protein kinase of extracellular signal-regulated kinase and cytokine production and secretion. The present genotyping assay was established in 61 subjects of Caucasian ethnicity and investigates the functional role of the selected genes in the context of the known genetic architecture of pain without seeking functional associations for pain. The assay identified a total of 691 genetic variants, of which many have reports for a clinical relevance for pain or in another context. The assay is applicable for small to large-scale experimental setups at contemporary genotyping costs

Computational Functional Genomics-Based AmpliSeq™ Panel for Next-Generation Sequencing of Key Genes of Pain

The genetic background of pain is becoming increasingly well understood, which opens up possibilities for predicting the individual risk of persistent pain and the use of tailored therapies adapted to the variant pattern of the patient’s pain-relevant genes. The individual variant pattern of pain-relevant genes is accessible via next-generation sequencing, although the analysis of all “pain genes” would be expensive. Here, we report on the development of a cost-effective next generation sequencing-based pain-genotyping assay comprising the development of a customized AmpliSeq™ panel and bioinformatics approaches that condensate the genetic information of pain by identifying the most representative genes. The panel includes 29 key genes that have been shown to cover 70% of the biological functions exerted by a list of 540 so-called “pain genes” derived from transgenic mice experiments. These were supplemented by 43 additional genes that had been independently proposed as relevant for persistent pain. The functional genomics covered by the resulting 72 genes is particularly represented by mitogen-activated protein kinase of extracellular signal-regulated kinase and cytokine production and secretion. The present genotyping assay was established in 61 subjects of Caucasian ethnicity and investigates the functional role of the selected genes in the context of the known genetic architecture of pain without seeking functional associations for pain. The assay identified a total of 691 genetic variants, of which many have reports for a clinical relevance for pain or in another context. The assay is applicable for small to large-scale experimental setups at contemporary genotyping costs

Computational Functional Genomics-Based AmpliSeq™ Panel for Next-Generation Sequencing of Key Genes of Pain

The genetic background of pain is becoming increasingly well understood, which opens up possibilities for predicting the individual risk of persistent pain and the use of tailored therapies adapted to the variant pattern of the patient’s pain-relevant genes. The individual variant pattern of pain-relevant genes is accessible via next-generation sequencing, although the analysis of all “pain genes” would be expensive. Here, we report on the development of a cost-effective next generation sequencing-based pain-genotyping assay comprising the development of a customized AmpliSeq™ panel and bioinformatics approaches that condensate the genetic information of pain by identifying the most representative genes. The panel includes 29 key genes that have been shown to cover 70% of the biological functions exerted by a list of 540 so-called “pain genes” derived from transgenic mice experiments. These were supplemented by 43 additional genes that had been independently proposed as relevant for persistent pain. The functional genomics covered by the resulting 72 genes is particularly represented by mitogen-activated protein kinase of extracellular signal-regulated kinase and cytokine production and secretion. The present genotyping assay was established in 61 subjects of Caucasian ethnicity and investigates the functional role of the selected genes in the context of the known genetic architecture of pain without seeking functional associations for pain. The assay identified a total of 691 genetic variants, of which many have reports for a clinical relevance for pain or in another context. The assay is applicable for small to large-scale experimental setups at contemporary genotyping costs

Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons

Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The calibre of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. In this study we quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (Dmean) in neurites from 0.12 ± 0.05 µm2/s to 0.61 ± 0.03 µm2/s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9 ± 1.9 to 15 ± 4.9 µm/s, whereas a velocity increase from 9 ± 1.3 to 14 ± 3 µm/s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons

Correlative Light- and Electron Microscopy with chemical tags

AbstractCorrelative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25nm in the x–y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells

Coordinate-based co-localization-mediated analysis of arrestin clustering upon stimulation of the C-C chemokine receptor 5 with RANTES/CCL5 analogues

G protein-coupled receptor activation and desensitization leads to recruitment of arrestin proteins from cytosolic pools to the cell membrane where they form clusters difficult to characterize due to their small size and further mediate receptor internalization. We quantitatively investigated clustering of arrestin 3 induced by potent anti-HIV analogues of the chemokine RANTES after stimulation of the C-C chemokine receptor 5 using single-molecule localization-based super-resolution microscopy. We determined arrestin 3 cluster sizes and relative fractions of arrestin 3 molecules in each cluster through image-based analysis of the localization data by adapting a method originally developed for co-localization analysis from molecular coordinates. We found that only classical agonists in the set of tested ligands were able to efficiently recruit arrestin 3 to clusters mostly larger than 150nm in size and compare our results with existing data on arrestin 2 clustering induced by the same chemokine analogues

Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells

Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls NF-ĸB (nuclear factor kappa-light-chain-enhancer of activated B-cells) signaling, cell proliferation, programmed cell death, and survival, and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor pre-dimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly into trimers but also into higher-order oligomers. A functional mutation in the pre-ligand assembly domain (PLAD) of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wildtype TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-ĸB signaling

Model-based identification of TNF alpha-induced IKK beta-mediated and I kappa B alpha-mediated regulation of NF kappa B signal transduction as a tool to quantify the impact of drug-induced liver injury compounds

Drug-induced liver injury: mathematical model quantifies impact of liver-damaging drugs Drug-induced liver injury (DILI) is one of the most important obstacles during drug development. More than 1000 drugs have been identified to damage the liver, but the current test systems are poor in predicting DILI. A team of cell biologists, theoretical physicists, and clinical pharmacologists combined experimental data generated in cultured liver cells with mathematical modeling to quantify the impact of the anti-inflammatory drug diclofenac. The analysis demonstrated that diclofenac induces multiple changes in the signal transduction network activated by the tumor necrosis factor alpha (TNFα), one of the known factors to amplify liver toxicity. Data of other liver injury-causing compounds were integrated into the mathematical model and their impact was quantified, thereby demonstrating the potential use of the mathematical model for the further analysis of other compounds in order to improve DILI test systems

Imaging-systems for localization-based super-resolution light-microscopy in physical biology : design and applications

Physical Biology is a field of life sciences dealing with the extraction of quantitative data from biophysical or molecular biological experiments with different levels of complexity. Such data are further used as parameters for mathematical models of the biological system. These models allow to predict reactions on external stimuli by describing the relevant molecular interactions and are therefore used for example to generate a deeper comprehension of complex human diseases. An essential technique in biophysical research on human diseases is fluorescence microscopy. This is a constantly developed toolbox comprising a large number of specific labeling strategies, as well as a broad spectrum of fluorescent probes. It is further minimal invasive and therefore suitable for measurements in living cells or organisms. The sensitivity of modern photo-detectors even allows for the detection of a single fluorescent probe with an accuracy of approximately 10 nm. ... The model-prediction was further verified by two color SMLM experiments. In this work the development and application of imaging-systems are described which provide quantitative data with single-molecule resolution for systems biological model approaches with a low degree of abstractness. In the near future, the impact of mathematical models in the research field of complex human diseases will increase. The predictions of these models will be more exact, the more detailed and accurate the input parameters will become. This work gives an impression of how quantitative data obtained by SMLM may serve as input parameters for mathematical models at the single-cell level.Die physikalische Biologie beschäftigt sich mit der Erhebung quantitativer Daten aus biophysikalischen und molekularbiologischen Experimenten unterschiedlicher Komplexität. Diese Daten können als Eingangsparameter für mathematische Modelle des zu untersuchenden biologischen Systems dienen. Solche systembiologischen Modelle bilden die relevanten Interaktionen zwischen Molekülen ab und erlauben das Verhalten des Systems auf externe Stimuli zu beschreiben. Ein essentieller Bestandteil biophysikalischer Untersuchungen von Zellen bis zu Organismen ist die Fluoreszenzmikroskopie. Sie zeichnet sich durch eine Vielzahl stetig weiterentwickelter, spezifischer Markierungsstrategien, sowie ein breites Spektrum an Fluoreszenzfarbstoffen aus. Durch eine geringe Invasivität ist sie geeignet, um Untersuchungen selbst an lebenden Zellen oder Organismen durchzuführen. Die hohe Sensitivität moderner Photosensoren erlaubt es selbst einzelne Fluoreszenzsonden mit einer Genauigkeit von ca. 10 nm zu detektieren. ... Die Arbeit beschreibt die Entwicklung und Anwendung von Einzelmolekül-Imaging-Systemen zur Erhebung quantitativer Daten auf Basis einzelner Zellen und mit der Auflösung einzelner Biomoleküle. Diese Daten dienen als Eingabeparameter für systembiologische Modelle von geringem Abstraktheitsgrad. In naher Zukunft werden systembiologische Modelle eine immer größere Rolle in der Erforschung humaner Erkrankungen finden. Die Modellvorhersagen werden mit steigender Genauigkeit der Eingabeparameter verlässlicher werden. Diese Arbeit gibt einen Einblick, welches Potential quantitative Daten aus Einzelmolekül-Imaging-Systemen als Eingabeparameter solcher Modelle inne haben