Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding. How these properties affect their ability to occupy specific genomic sites and modify the epigenetic landscape is unclear. The association of TFs with mitotic chromosomes observed by fluorescence microscopy is largely mediated by non-specific DNA interactions and differs broadly between TFs. Here we combine quantitative measurements of mitotic chromosome binding (MCB) of 501 TFs, TF mobility measurements by fluorescence recovery after photobleaching, single molecule imaging of DNA binding, and mapping of TF binding and chromatin accessibility. TFs associating to mitotic chromosomes are enriched in DNA-rich compartments in interphase and display slower mobility in interphase and mitosis. Remarkably, MCB correlates with relative TF on-rates and genome-wide specific site occupancy, but not with TF residence times. This suggests that non-specific DNA binding properties of TFs regulate their search efficiency and occupancy of specific genomic sites.

PurposeThe purpose of this paper is to describe the pre‐design and sizing of a smart leading edge section which is developed in the project SADE (Smart High Lift Devices for Next Generation Wings), which is part of the seventh framework program of the EU.Design/methodology/approachThe development of morphing technologies in SADE concentrates on the leading and trailing edge high‐lift devices. At the leading edge a smart gap and step‐less droop nose device is developed. For the landing flap a smart trailing edge of the flap is in the focus of the research activities. The main path in SADE follows the development of the leading edge section and the subsequent wind tunnel testing of a five meter span full‐scale section with a chord length of three meters in the wind tunnel T‐101 at the Russian central aero‐hydrodynamic institute (TsAGI) in Moscow.FindingsThe presented paper gives an overview over the desired performance and requirements of a smart leading edge device, its aerodynamic design for the wind tunnel tests and the structural pre‐design and sizing of the full‐scale leading edge section which will be tested in the wind tunnel.Originality/valueSADE aims at a major step forward in the development and evaluation of the potential of morphing airframe technologies.

Imaging, tracking and analyzing individual biomolecules in living systems is a powerful technology to obtain quantitative kinetic and spatial information such as reaction rates, diffusion coefficients and localization maps. Common tracking tools often operate on single movies and require additional manual steps to analyze whole data sets or to compare different experimental conditions. We report a fast and comprehensive single molecule tracking and analysis framework (TrackIt) to simultaneously process several multi-movie data sets. A user-friendly GUI offers convenient tracking visualization, multiple state-of-the-art analysis procedures, display of results, and data im- and export at different levels to utilize external software tools. We applied our framework to quantify dissociation rates of a transcription factor in the nucleus and found that tracking errors, similar to fluorophore photobleaching, have to be considered for reliable analysis. Accordingly, we developed an algorithm, which accounts for both tracking losses and suggests optimized tracking parameters when evaluating reaction rates. Our versatile and extensible framework facilitates quantitative analysis of single molecule experiments at different experimental conditions.

Actions of molecular species, for example binding of transcription factors to chromatin, may comprise several superimposed reaction pathways. The number and the rate constants of such superimposed reactions can in principle be resolved by inverse Laplace transformation of the corresponding distribution of reaction lifetimes. However, current approaches to solve this transformation are challenged by photobleaching-prone fluorescence measurements of lifetime distributions. Here, we present a genuine rate identification method (GRID), which infers the quantity, rates and amplitudes of dissociation processes from fluorescence lifetime distributions using a dense grid of possible decay rates. In contrast to common multi-exponential analysis of lifetime distributions, GRID is able to distinguish between broad and narrow clusters of decay rates. We validate GRID by simulations and apply it to CDX2-chromatin interactions measured by live cell single molecule fluorescence microscopy. GRID reveals well-separated narrow decay rate clusters of CDX2, in part overlooked by multiexponential analysis. We discuss the amplitudes of the decay rate spectrum in terms of frequency of observed events and occupation probability of reaction states. We further demonstrate that a narrow decay rate cluster is compatible with a common model of TF sliding on DNA.The actions of biomolecules are governed by thermal fluctuations and thus are intrinsically stochastic. Accordingly, interactions such as association and dissociation events of molecular species often follow Poissonian statistics with a constant probability per time, the rate constant, to occur. In this case, the experimentally accessible lifetime of the reaction is exponentially distributed. Commonly, a biomolecule engages in several different types of interaction, with each interaction type having its own reaction rate. For example, a biomolecule might bind to different protein species, to multiple sites on DNA or RNA, or to different cellular compartments. In such a scenario, not all members of a biomolecular specie will undergo the same type of interaction at any time. Rather, each biomolecule will conduct one of the multiple possible types of interaction. If the measurement determining the reaction lifetimes cannot distinguish between the different types of interaction, the resulting lifetime distribution will be multi-exponential and include reaction rates from all superimposed Poisson processes. More precisely, the lifetime distribution is a Laplace transform of the spectrum of reaction rates inherent to the biomolecule (Fig. 1a). Retrieving the underlying spectrum of reaction rates consequently evokes an inverse Laplace transformation.The inverse Laplace transformation is an ill-posed problem for the inversion of inherently noisy, discrete distributions and numerical solutions are often unstable 1,2 . Nevertheless, algorithms treating the Laplace transform using gradient methods and appropriate regularization have been successfully developed for noisy data in NMR 3,4 and prote...

Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequencespecific/non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape.