Following Toll-like receptor 4 (TLR4) stimulation of macrophages, negative feedback mediated by the anti-inflammatory cytokine interleukin-10 (IL-10) limits the inflammatory response. However, ...extensive cell-to-cell variability in TLR4-stimulated cytokine secretion raises questions about how negative feedback is robustly implemented. To explore this, we characterize the TLR4-stimulated secretion program in primary murine macrophages using a single-cell microwell assay that enables evaluation of functional autocrine IL-10 signaling. High-dimensional analysis of single-cell data reveals three tiers of TLR4-induced proinflammatory activation based on levels of cytokine secretion. Surprisingly, while IL-10 inhibits TLR4-induced activation in the highest tier, it also contributes to the TLR4-induced activation threshold by regulating which cells transition from non-secreting to secreting states. This role for IL-10 in restraining TLR4 inflammatory activation is largely mediated by intermediate interferon (IFN)-β signaling, while TNF likely mediates response resolution by IL-10. Thus, cell-to-cell variability in cytokine regulatory motifs provides a means to tailor the TLR4-induced inflammatory response.
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•TLR4 activation in macrophage populations is resolved by IL-10 negative feedback•IL-10 secretion is decoupled from TLR4 proinflammatory activation in isolated cells•IL-10 activity contributes to a threshold that restrains TLR4-induced activation•IL-10’s role in restraining TLR4 activation is largely mediated by IFN-β signaling
Alexander et al. use multiplexed single-cell secretion analysis to disentangle TLR4-induced autocrine feedback loops. They show that IL-10 raises the threshold of inflammatory activation in macrophages while also negatively regulating highly activated cells. The restraining role of IL-10 is mediated by IFN-β signaling, while TNF-induced IL-10 likely mediates response resolution.
Highly multiplexed detection of proteins secreted by single cells is always challenging. Herein, a multiplexed in situ tagging technique based on single‐stranded DNA encoded microbead arrays and ...multicolor successive imaging for assaying single‐cell secreted proteins with high throughput and high sensitivity is presented. This technology is demonstrated to be capable of increasing the multiplexity exponentially. Upon integration with polydimethylsiloxane microwells, this platform is applied to detect ten immune effector proteins from differentiated single macrophages stimulated with lipopolysaccharide. Significant heterogeneity is observed when the derived human primary macrophages are analyzed. This versatile technology is expected to open new opportunities in systems biology, immune regulation studies, signaling analysis, and molecular diagnostics.
A multiplexed in situ tagging technique based on single‐stranded DNA encoded microbead arrays and multicolor successive imaging is developed for single‐cell secreted protein detection. This technology is demonstrated to be reliable and capable of increasing the multiplexity exponentially. This versatile platform is expected to open new opportunities in systems biology, immune regulation studies, signaling analysis, and molecular diagnostics.
Targeting nanoparticles as drug delivery platforms is crucial to facilitate their cellular entry. Docking of nanoparticles by targeting ligands on cell membranes is the first step for the initiation ...of cellular uptake. As a model system, we studied brain microvascular endothelial cells, which form the anatomical basis of the blood–brain barrier, and the tripeptide glutathione, one of the most effective targeting ligands of nanoparticles to cross the blood–brain barrier. To investigate this initial docking step between glutathione and the membrane of living brain endothelial cells, we applied our recently developed innovative optical method. We present a microtool, with a task-specific geometry used as a probe, actuated by multifocus optical tweezers to characterize the adhesion probability and strength of glutathione-coated surfaces to the cell membrane of endothelial cells. The binding probability of the glutathione-coated surface and the adhesion force between the microtool and cell membrane was measured in a novel arrangement: cells were cultured on a vertical polymer wall and the mechanical forces were generated laterally and at the same time, perpendicularly to the plasma membrane. The adhesion force values were also determined with more conventional atomic force microscopy (AFM) measurements using functionalized colloidal probes. The optical trapping-based method was found to be suitable to measure very low adhesion forces (≤ 20 pN) without a high level of noise, which is characteristic for AFM measurements in this range. The holographic optical tweezers-directed functionalized microtools may help characterize the adhesion step of nanoparticles initiating transcytosis and select ligands to target nanoparticles.
•High nitrogen concentrations have a negative effect on radial growth.•Overfertilized trees produce tracheids with thinner cell walls.•Wood structure in over-fertilized trees can affect water ...transport in the trunk.•Overfertilized trees produce wood with inferior wood properties.
Fertilisation is often used to increase plant productivity in agriculture but has also been used in forestry. In our study, Scots pine forest growing in a nitrogen-poor environment was fertilised with NPK post-production wastewater from a potato starch factory. Our research aimed to investigate the dependence of tree growth on different NPK concentrations. Cell characteristics such as cell wall thickness (CWT), lumen diameter (LD) and tree-ring features such as ring width (RW), total number of cells in annual growth (nTotal), earlywood (EW) and latewood (LW) were investigated. Twenty-six years of regular fertilisation of the forest with different doses of wastewater rich in NPK elements have affected the anatomical structure of Scots pine trees. It is presumed that the reduction in CWT and LD on the fertilised site was due to deficiencies in plant water conductivity, which may have occurred due to physiological drought. The influence of nitrogen on unfertilised site from the wastewater area could contribute to the CWT thickening. The results confirm that the use of NPK in excessive doses is detrimental to trees' conductive system.
Protein-binding dynamics imaged in a living cell Phillip, Yael; Kiss, Vladimir; Schreiber, Gideon
Proceedings of the National Academy of Sciences - PNAS,
01/2012, Volume:
109, Issue:
5
Journal Article
Peer reviewed
Open access
Historically, rate constants were determined in vitro and it was unknown whether they were valid for in vivo biological processes. Here, we bridge this gap by measuring binding dynamics between a ...pair of proteins in living HeLa cells. Binding of a β-lactamase to its protein inhibitor was initiated by microinjection and monitored by Förster resonance energy transfer. Association rate constants for the wild-type and an electrostatically optimized mutant were only 25% and 50% lower than in vitro values, whereas no change in the rate constant was observed for a slower binding mutant. These changes are much smaller than might be anticipated considering the high macromolecular crowding within the cell. Single-cell analyses of association rate constants and fluorescence recovery after photobleaching reveals a naturally occurring variation in cell density, which is translated to an up to a twofold effect on binding rate constants. The data show that for this model protein interaction the intracellular environment had only a small effect on the association kinetics, justifying the extrapolation of in vitro data to processes in the cell.
Nutrient limitation is one of the most common triggers of antibiotic tolerance and persistence. Here, we present two microfluidic setups to study how spatial and temporal variation in nutrient ...availability lead to increased survival of bacteria to antibiotics. The first setup is designed to mimic the growth dynamics of bacteria in spatially structured populations (e.g., biofilms) and can be used to study how spatial gradients in nutrient availability, created by the collective metabolic activity of a population, increase antibiotic tolerance. The second setup captures the dynamics of feast-and-famine cycles that bacteria recurrently encounter in nature, and can be used to study how phenotypic heterogeneity in growth resumption after starvation increases survival of clonal bacterial populations. In both setups, the growth rates and metabolic activity of bacteria can be measured at the single-cell level. This is useful to build a mechanistic understanding of how spatiotemporal variation in nutrient availability triggers bacteria to enter phenotypic states that increase their tolerance to antibiotics.
Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length ...scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of theArabidopsisecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifierATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall.
Cell adhesion is a protein-mediated process intrinsic to most living organisms. Dysfunction in cell adhesion processes is implicated in various diseases, including thrombosis and metastatic cancers. ...Using an approach to resolve spectral features from cell membrane-associated photoluminescent single-walled carbon nanotubes, we found that nanotube optical bandgaps respond to the electrostatic potential of the cell surface, which corresponds to cell adhesion properties. We studied the carbon nanotube emission energy response to solution ionic potentials, which suggests sensitivity to local charge accumulation. We conclude that nanotubes respond to cell surface electrostatic potentials that are mediated by membrane proteins, which vary significantly across cell types. These findings portend the optical measurement of surface electrostatic potentials for biophysical measurements and biomedical applications.
Distinct differences between how model proteins interact in-cell and in vitro suggest that the cytosol might have a profound effect in modulating protein-protein and/or protein-ligand interactions ...that are not observed in vitro. Analyses of in-cell NMR spectra of target proteins interacting with physiological partners are further complicated by low signal-to-noise ratios, and the long overexpression times used in protein-protein interaction studies may lead to changes in the in-cell spectra over the course of the experiment. To unambiguously resolve the principal binding mode between two interacting species against the dynamic cellular background, we analyzed in-cell spectral data of a target protein over the time course of overexpression of its interacting partner by using single-value decomposition (SVD). SVD differentiates between concentration-dependent and concentration-independent events and identifies the principal binding mode between the two species. The analysis implicates a set of amino acids involved in the specific interaction that differs from previous NMR analyses but is in good agreement with crystallographic data.
Precise gene expression patterns are established by transcription factor (TFs) binding to regulatory sequences. While these events occur in the context of chromatin, our understanding of how ...TF-nucleosome interplay affects gene expression is highly limited. Here, we present an assay for high-resolution measurements of both DNA occupancy and gene expression on large-scale libraries of systematically designed regulatory sequences. Our assay reveals occupancy patterns at the single-cell level. It provides an accurate quantification of the fraction of the population bound by a nucleosome and captures distinct, even adjacent, TF binding events. By applying this assay to over 1,500 promoter variants in yeast, we reveal pronounced differences in the dependency of TF activity on chromatin and classify TFs by their differential capacity to alter chromatin and promote expression. We further demonstrate how different regulatory sequences give rise to nucleosome-mediated TF collaborations that quantitatively account for the resulting expression.
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•Parallel expression and occupancy measurements reveal TF-nucleosome interplay•Pronounced differences in transcription factors (TFs) sensitivity to chromatin•Specific combinations of TF sites give rise to nucleosome-mediated collaborations•Properties of nucleosome-based collaborations can quantitatively shape expression
With parallel measurements of gene expression and DNA occupancy, Levo et al. examine transcription factors (TFs) activity in the context of chromatin. Regulatory sequences, with various combinations and arrangements of TF sites, give rise to different nucleosome-mediated collaborations between TFs. These, in turn, can quantitatively account for the resulting expression.