Strain-induced alignment in collagen gels

Collagen is the most abundant extracellular-network-forming protein in animal biology and is important in both natural and artificial tissues, where it serves as a material of great mechanical versatility. This versatility arises from its almost unique ability to remodel under applied loads into anisotropic and inhomogeneous structures. To explore the origins of this property, we develop a set of analysis tools and a novel experimental setup that probes the mechanical response of fibrous networks in a geometry that mimics a typical deformation profile imposed by cells in vivo. We observe strong fiber alignment and densification as a function of applied strain for both uncrosslinked and crosslinked collagenous networks. This alignment is found to be irreversibly imprinted in uncrosslinked collagen networks, suggesting a simple mechanism for tissue organization at the microscale. However, crosslinked networks display similar fiber alignment and the same geometrical properties as uncrosslinked gels, but with full reversibility. Plasticity is therefore not required to align fibers. On the contrary, our data show that this effect is part of the fundamental non-linear properties of fibrous biological networks.Comment: 12 pages, 7 figures. 1 supporting material PDF with 2 figure

News from the Pizza-Connection

Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown, although it is important for gene regulation and replication. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal structure, except during cell division. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in interphase. Two models for the detailed folding of the 30 nm chromatin fiber within these territories are under debate: In the Random- Walk/Giant-Loop-model big loops of 3 to 5 Mbp are attached to a non-DNA backbone. In the Multi-Loop- Subcompartment (MLS) model loops of around 120 kbp are forming rosettes, which are also interconnected by the chromatin fiber. Here we show with a comparison between simulations and experiments an interdisciplinary approach leading to a determination of the three-dimensional organization of the human genome: For the predictions of experiments various models of human interphase chromosomes and the whole cell nucleus were simulated with Monte Carlo and Brownian Dynamics methods. Only the MLS-model leads to the formation of non-overlapping chromosome territories and distinct functional and dynamic subcompartments in agreement with experiments. Fluorescernce in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labeling is visualized with confocal laser scanning microscopy followed by image reconstruction procedures. Chromosome arms show only small overlap and globular substructures as predicted by the MLS-model. The spatial distances between pairs of genomic markers as function of their genomic separation result in a MLS-model with loop and linker sizes around 126 kbp. With the development of GFP-fusion-proteins it is possible to study the chromatin distribution and dynamics resulting from cell cycle, treatment by chemicals or radiation in vivo. The chromatin distributions are similar to those found in the simulation of whole cell nuclei of the MLS-model. Fractal analysis is especially suited to quantify the unordered and non-euklidean chromatin distribution of the nucleus. The dynamic behaveour of the chromatin structure and the diffusion of particles in the nucleus are also closely connected to the fractal dimension. Fractal analysis of the simulations reveal the multi-fractality of chromosomes. First fractal analysis of chromatin distributions in vivo result in significant differences for different morphologies and might favour a MLS-model- like chromatin distribution. Simulations of fragment distributions based on double strand breakage after carbon- ion irradiation differ in different models. Here again a comparison with experiments favours a MLS-model

Binding-Site Assessment by Virtual Fragment Screening

The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules) would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock ∼11000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors

Three-dimensional organization of chromosome territories in the human interphase cell nucleus.

The synthesis of proteins, maintenance of structure and duplication of the eukaryotic cell itself are all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. The regulation of genes – their transcription and replication - has been shown to be connected closely to the threedimensional organization of the genome in the cell nucleus. Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown. The nucleus of the cell has for a long time been viewed as a 'spaghetti soup' of DNA bound to various proteins without much internal structure, except during cell division when chromosomes are condensed into separate entities. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in the interphase, i.e. between cell divisions. In an analogy of the Bauhaus principle that "form follows function" we believe that analyzing in which form DNA is organized in these territories will help us to understand genomic function. We use computer models - Monte Carlo and Brownian dynamics simulations - to develop plausible proposals for the structure of the interphase genome and compare them to experimental data. In the work presented here, we simulate interphase chromosomes for different folding morphologies of the chromatin fiber which is organized into loops of 100kbp to 3 Mbp that can be interconnected in various ways. The backbone of the fiber is described by a wormlike-chain polymer whose diameter and stiffness can be estimated from independent measurements. The implementation describes this polymer as a segmented chain with 3000 to 20000 segments for chromosome 15 depending on the phase of the simulation. The modeling is performed on a parallel computer (IBM SP2 with 80 nodes). We also determine genomic marker distributions within the Prader- Willi-Region on chromosome 15q11.2-13.3. For these measurements we use a fluorescence in situ hybridisation method (in collaboration with I. Solovai, J. Craig and T. Cremer, Munich, FRG) conserving the structure of the nucleus. As probes we use 10 kbp long lambda clones (Prof. B. Horsthemke, Essen, FRG) covering genomic marker distances between 8 kbp and 250 kbp. The markers are detected with confocal and standing wavefield light microscopes (in collaboration with J.Rauch, J. Bradl, C. Cremer and E.Stelzer, both Heidelberg, FRG) and using special image reconstruction methods developed solely for this purpose (developed by R. Eils. and W. Jaeger, Heidelberg, FRG). Best agreement between simulations and experiments is reached for a Multi-Loop-Subcompartment model with a loop size of 126 kbp which are forming rosetts and are linked by a chromatin linker of 126 kbp. We also hypothesize a different folding structure for maternal versus paternal chromosome 15. In simulations of whole cell nuclei this modell also leads to distinct chromosome territories and subcompartments. A fractal analysis of the simulations leads to multifractal behavior in good agreement with predictions drawn from porous network research

Estimating Absolute Configurational Entropies of Macromolecules: The Minimally Coupled Subspace Approach

We develop a general minimally coupled subspace approach (MCSA) to compute absolute entropies of macromolecules, such as proteins, from computer generated canonical ensembles. Our approach overcomes limitations of current estimates such as the quasi-harmonic approximation which neglects non-linear and higher-order correlations as well as multi-minima characteristics of protein energy landscapes. Here, Full Correlation Analysis, adaptive kernel density estimation, and mutual information expansions are combined and high accuracy is demonstrated for a number of test systems ranging from alkanes to a 14 residue peptide. We further computed the configurational entropy for the full 67-residue cofactor of the TATA box binding protein illustrating that MCSA yields improved results also for large macromolecular systems

Three-dimensional organization of chromosome territories in the human interphase nucleus

The synthesis of proteins, maintenance of structure and duplication of the eukaryotic cell itself are all fine-tuned biochemical processes that depend on the precise structural arrangement of the cellular components. The regulation of genes – their transcription and replication - has been shown to be connected closely to the three- dimensional organization of the genome in the cell nucleus. Despite the successful linear sequencing of the human genome its three-dimensional structure is widely unknown. The nucleus of the cell has for a long time been viewed as a 'spaghetti soup' of DNA bound to various proteins without much internal structure, except during cell division when chromosomes are condensed into separate entities. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in the interphase, i.e. between cell divisions. In an analogy of the Bauhaus principle that "form follows function" we believe that analyzing in which form DNA is organized in these territories will help us to understand genomic function. We use computer models - Monte Carlo and Brownian dynamics simulations - to develop plausible proposals for the structure of the interphase genome and compare them to experimental data. In the work presented here, we simulate interphase chromosomes for different folding morphologies of the chromatin fiber which is organized into loops of 100kbp to 3 Mbp that can be interconnected in various ways. The backbone of the fiber is described by a wormlike-chain polymer whose diameter and stiffness can be estimated from independent measurements. The implementation describes this polymer as a segmented chain with 3000 to 20000 segments for chromosome 15 depending on the phase of the simulation. The modeling is performed on a parallel computer (IBM SP2 with 80 nodes). We also determine genomic marker distributions within the Prader- Willi-Region on chromosome 15q11.2-13.3. For these measurements we use a fluorescence in situ hybridisation method (in collaboration with I. Solovai, J. Craig and T. Cremer, Munich, FRG) conserving the structure of the nucleus. As probes we use 10 kbp long lambda clones (Prof. B. Horsthemke, Essen, FRG) covering genomic marker distances between 8 kbp and 250 kbp. The markers are detected with confocal and standing wavefield light microscopes (in collaboration with J.Rauch, J. Bradl, C. Cremer and E.Stelzer, both Heidelberg, FRG) and using special image reconstruction methods developed solely for this purpose (developed by R. Eils. and W. Jaeger, Heidelberg, FRG). Best agreement between simulations and experiments is reached for a Multi-Loop-Subcompartment model with a loop size of 126 kbp which are forming rosetts and are linked by a chromatin linker of 126 kbp. We also hypothesize a different folding structure for maternal versus paternal chromosome 15. In simulations of whole cell nuclei this modell also leads to distinct chromosome territories and subcompartments. A fractal analysis of the simulations leads to multifractal behavior in good agreement with predictions drawn from porous network research

Facilitated diffusion of DNA-binding proteins

The diffusion-controlled limit of reaction times for site-specific DNA-binding proteins is derived from first principles. We follow the generally accepted concept that a protein propagates via two competitive modes, a three-dimensional diffusion in space and a one-dimensional sliding along the DNA. However, our theoretical treatment of the problem is new. The accuracy of our analytical model is verified by numerical simulations. The results confirm that the unspecific binding of protein to DNA, combined with sliding, is capable to reduce the reaction times significantly.Comment: 4 pages, 2 figures Nov 22 2005 - accepted for PR

Modelling diffusional transport in the interphase cell nucleus

In this paper a lattice model for diffusional transport of particles in the interphase cell nucleus is proposed. Dense networks of chromatin fibers are created by three different methods: randomly distributed, non-interconnected obstacles, a random walk chain model, and a self avoiding random walk chain model with persistence length. By comparing a discrete and a continuous version of the random walk chain model, we demonstrate that lattice discretization does not alter particle diffusion. The influence of the 3D geometry of the fiber network on the particle diffusion is investigated in detail, while varying occupation volume, chain length, persistence length and walker size. It is shown that adjacency of the monomers, the excluded volume effect incorporated in the self avoiding random walk model, and, to a lesser extent, the persistence length, affect particle diffusion. It is demonstrated how the introduction of the effective chain occupancy, which is a convolution of the geometric chain volume with the walker size, eliminates the conformational effects of the network on the diffusion, i.e., when plotting the diffusion coefficient as a function of the effective chain volume, the data fall onto a master curve.Comment: 9 pages, 8 figure

Facilitated diffusion of DNA-binding proteins: Simulation of large systems

The recently introduced method of excess collisions (MEC) is modified to estimate diffusion-controlled reaction times inside systems of arbitrary size. The resulting MEC-E equations contain a set of empirical parameters, which have to be calibrated in numerical simulations inside a test system of moderate size. Once this is done, reaction times of systems of arbitrary dimensions are derived by extrapolation, with an accuracy of 10 to 15 percent. The achieved speed up, when compared to explicit simulations of the reaction process, is increasing proportional to the extrapolated volume of the cell.Comment: 8 pages, 4 figures, submitted to J. Chem. Phy