Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without ...perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.
The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire ...mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.
Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform ...multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.
Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of PRC2 function ...correlates with certain malignancies and poor prognosis. EZH2 is the catalytic engine of the PRC2 complex and thus represents a key candidate oncology target for pharmacological intervention. Here we report the optimization of our indole-based EZH2 inhibitor series that led to the identification of CPI-1205, a highly potent (biochemical IC50 = 0.002 μM, cellular EC50 = 0.032 μM) and selective inhibitor of EZH2. This compound demonstrates robust antitumor effects in a Karpas-422 xenograft model when dosed at 160 mg/kg BID and is currently in Phase I clinical trials. Additionally, we disclose the co-crystal structure of our inhibitor series bound to the human PRC2 complex.
The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution ...and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P
and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P
, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP-WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P
, and PI(3)P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P
alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.
The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) is important for the initiation of allergic airway inflammation through a dendritic cell-mediated T helper 2 response. To identify ...the factors that control TSLP expression, we examined the ability of inflammatory mediators to regulate TSLP production in human airway epithelial cells. We found that both IL-1β and TNF-α were capable of inducing rapid TSLP production in primary human bronchial airway epithelial cells. We further characterized the human TSLP gene promoter, using two human epithelial cell lines, 16HBEo⁻ and A549, and showed that IL-1β- and TNF-α-mediated human TSLP promoter activation in these cells was mediated by an upstream NFκB site. Mutation of this NFκB site abolished activation, as did overexpression of a dominant-negative version of IκB kinase (IKK)β (a kinase acting on IκB, the inhibitor of NFκB). Interestingly, human TSLP mRNA levels were also increased after exposure to Toll-like receptor (TLR) 2, TLR8, and TLR9 ligands, further supporting an important role for NFκB in TSLP gene regulation. Similarly, analysis of the mouse TSLP gene promoter revealed the presence of a similarly situated NFκB site that was also critical for IL-1β-inducible expression of mouse TSLP. Taken together, these results demonstrate that the inflammatory mediators IL-1β and TNF-α regulate human TSLP gene expression in an NFκB-dependent manner.
Background Respiratory viral infection, including respiratory syncytial virus (RSV) and rhinovirus, has been linked to respiratory disease in pediatric patients, including severe acute bronchiolitis ...and asthma exacerbation. Objective The study examined the role of the epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) in the response to RSV infection. Methods Infection of human airway epithelial cells was used to examine TSLP induction after RSV infection. Air–liquid interface cultures from healthy children and children with asthma were also tested for TSLP production after infection. Finally, a mouse model was used to directly test the role of TSLP signaling in the response to RSV infection. Results Infection of airway epithelial cells with RSV led to the production of TSLP via activation of an innate signaling pathway that involved retinoic acid induced gene I, interferon promoter-stimulating factor 1, and nuclear factor-κB. Consistent with this observation, airway epithelial cells from asthmatic children a produced significantly greater levels of TSLP after RSV infection than cells from healthy children. In mouse models, RSV-induced TSLP expression was found to be critical for the development of immunopathology. Conclusion These findings suggest that RSV can use an innate antiviral signaling pathway to drive a potentially nonproductive immune response and has important implications for the role of TSLP in viral immune responses in general.
The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic inflammation in both humans and mice. TSLP has been shown to promote a Th2‐type ...response through upregulation of OX40L on dendritic cells, and through direct induction of IL‐4 production in naïve CD4+ T cells. However, its direct effect on effector Th cells has not been extensively investigated. In this study, we show that the level of TSLP receptor (TSLPR) expression on mouse effector Th2 cells is higher than on Th1 and Th17 cells, and that TSLP induced proliferation of effector Th2, but not Th1 nor Th17 cells. TSLP also induced the phosphorylation of signal transducer and activator of transcription (Stat) 5, and expression of the anti‐apoptotic factor Bcl‐2 in Th2 cells. Finally, TSLP‐mediated proliferation on Th2 cells was enhanced by TCR stimulation, through IL‐4‐mediated induction of TSLPR expression. Taken together, these results indicate that TSLP is involved in exacerbation of mouse Th2‐mediated allergic inflammation in a Th2 environment through direct stimulation of Th2 effector cells.
Hexagonal boron nitride (h-BN) is a 2D, wide band gap semiconductor that has recently been shown to display bright room-temperature emission in the visible region, sparking immense interest in the ...material for use in quantum applications. In this work, we study highly crystalline, single atomic layers of chemical vapor deposition grown h-BN and find predominantly one type of emissive state. Using a multidimensional super-resolution fluorescence microscopy technique we simultaneously measure spatial position, intensity, and spectral properties of the emitters, as they are exposed to continuous wave illumination over minutes. As well as low emitter heterogeneity, we observe inhomogeneous broadening of emitter line-widths and power law dependency in fluorescence intermittency; this is strikingly similar to previous work on quantum dots. These results show that high control over h-BN growth and treatment can produce a narrow distribution of emitter type and that surface interactions heavily influence the photodynamics. Furthermore, we highlight the utility of spectrally resolved wide-field microscopy in the study of optically active excitations in atomically thin two-dimensional materials.
Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe’s diffraction limit, extending the frontier of noninvasive ...imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20–40 nm in 3D) over a large field of view (12 {hebrewnun}12 μm).
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