Mechanics of the IL2RA Gene Activation Revealed by Modeling and Atomic Force Microscopy

Transcription implies recruitment of RNA polymerase II and transcription factors (TFs) by DNA melting near transcription start site (TSS). Combining atomic force microscopy and computer modeling, we investigate the structural and dynamical properties of the IL2RA promoter and identify an intrinsically negative supercoil in the PRRII region (containing Elf-1 and HMGA1 binding sites), located upstream of a curved DNA region encompassing TSS. Conformational changes, evidenced by time-lapse studies, result in the progressive positioning of curvature apex towards the TSS, likely facilitating local DNA melting. In vitro assays confirm specific binding of the General Transcription Factors (GTFs) TBP and TFIIB over TATA-TSS position, where an inhibitory nucleosome prevented preinitiation complex (PIC) formation and uncontrolled DNA melting. These findings represent a substantial advance showing, first, that the structural properties of the IL2RA promoter are encoded in the DNA sequence and second, that during the initiation process DNA conformation is dynamic and not static

Single-nanotube tracking reveals the nanoscale organization of the extracellular space in the live brain

This work was supported by CNRS, the Agence Nationale de la Recherche (ANR-14-OHRI-0001-01), IdEx Bordeaux (ANR-10-IDEX-03-02), Labex Brain (ANR-10-LABX-43), Conseil Régional d'Aquitaine (2011-1603009) and the France-BioImaging national infrastructure (ANR-10-INBS-04-01). A.G.G. acknowledges financial support from the Fondation pour la Recherche Médicale and the Fonds Recherche du Québec–Nature et Technologies. J.A.V. acknowledges funding from Marie Curie Individual Fellowship 326442.The brain is a dynamic structure with the extracellular space (ECS) taking up almost a quarter of its volume. Signalling molecules, neurotransmitters and nutrients transit via the ECS, which constitutes a key microenvironment for cellular communication and the clearance of toxic metabolites. The spatial organization of the ECS varies during sleep, development and aging and is probably altered in neuropsychiatric and degenerative diseases, as inferred from electron microscopy and macroscopic biophysical investigations. Here we show an approach to directly observe the local ECS structures and rheology in brain tissue using super-resolution imaging. We inject single-walled carbon nanotubes into rat cerebroventricles and follow the near-infrared emission of individual nanotubes as they diffuse inside the ECS for tens of minutes in acute slices. Because of the interplay between the nanotube geometry and the ECS local environment, we can extract information about the dimensions and local viscosity of the ECS. We find a striking diversity of ECS dimensions down to 40 nm, and as well as of local viscosity values. Moreover, by chemically altering the extracellular matrix of the brains of live animals before nanotube injection, we reveal that the rheological properties of the ECS are affected, but these alterations are local and inhomogeneous at the nanoscale.PostprintPeer reviewe

Detection of single-molecule H₂O₂ signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes

An emerging concept in cell signalling is the natural role of reactive oxygen species such as hydrogen peroxide (H₂O₂) as beneficial messengers in redox signalling pathways. The nature of H₂O₂ signalling is confounded, however, by difficulties in tracking it in living systems, both spatially and temporally, at low concentrations. Here, we develop an array of fluorescent single-walled carbon nanotubes that can selectively record, in real time, the discrete, stochastic quenching events that occur as H₂O₂molecules are emitted from individual human epidermal carcinoma cells stimulated by epidermal growth factor. We show mathematically that such arrays can distinguish between molecules originating locally on the cell membrane from other contributions. We find that epidermal growth factor induces 2 nmol H₂O₂ locally over a period of 50 min. This platform promises a new approach to understanding the signalling of reactive oxygen species at the cellular level.Arnold and Mabel Beckman Foundation (Beckman Young Investigator award)National Science Foundation (U.S.) ((NSF) Career Award)National Science Foundation (U.S.) (Nanoscale Interdisciplinary Research Team)Czech Republic. Ministry of Education, Youth, and Sports (project no. MSM0021620806)Czech Republic. Ministry of Education, Youth, and Sports (KAN grant no. 400100701