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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2015

Thermal motion of DNA in an MspA pore

We report on an experiment and calculations that determine the thermal motion of a voltage-clamped ssDNA-NeutrAvidin complex in an MspA nanopore. The electric force and diffusion constant of DNA inside an MspA pore have been determined in order to evaluate DNAs thermal position fluctuations. We show that an out-of-equilibrium state returns to equilibrium so quickly that experiments usually measure a weighted average over the equilibrium position distribution. Averaging over the equilibrium position distribution is consistent with results of state-of-the-art nanopore sequencing experiments. It is shown that a reduction in thermal averaging can be achieved by increasing the electrophoretic force used in nanopore sequencing devices.

Biophysics↗

bioRxiv2015

Gctf: real-time CTF determination and correction

Accurate estimation of the contrast transfer function (CTF) is critical for a near-atomic resolution cryo electron microscopy (cryoEM) reconstruction. Here, I present a GPU-accelerated computer program, Gctf, for accurate and robust, real-time CTF determination. Similar to alternative programs, the main target of Gctf is to maximize the cross-correlation of a simulated CTF with the power spectra of observed micrographs after background reduction. However, novel approaches in Gctf improve both speed and accuracy. In addition to GPU acceleration, a fast 1-dimensional search plus 2-dimensional refinement (1S2R) procedure significantly speeds up Gctf. Based on the global CTF determination, the local defocus for each particle and for single frames of movies is accurately refined, which improves CTF parameters of all particles for subsequent image processing. Novel diagnosis method using equiphase averaging(EFA) and self-consistency verification procedures have also been implemented in the program for practical use, especially for aims of near-atomic reconstruction. Gctf is an independent program and the outputs can be easily imported into other cryoEM software such as Relion and Frealign. The results from several representative datasets are shown and discussed in this paper.

Biophysics↗

bioRxiv2015

Super-resolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes

During meiosis, homologous chromosomes associate to form a unique structure called synaptonemal complex (SC) whose disruption leads to infertility. Information about the epigenetic features of chromatin within this structure at the level of super-resolution microscopy is largely lacking. We combined single molecule localization microscopy with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be non-randomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin (trimethylation of histone H3 at lysine 27, H3K27me3) are found at 500 nm intervals along the SC, while one of the ends of SC displays a large and dense cluster of centromeric histone mark (trimethylation of histone H3 at lysine 9, H3K9me3). Chromatin associated with active transcription (trimethylation of histone H3 at lysine 4, H3K4me3) is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 mark with an average spread larger than their spacing. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher-order genome organisation is yet to be elucidated.

Biophysics↗

bioRxiv2015

Modeling the overproduction of ribosomes when antibacterial drugs act on cells

Bacteria that are subjected to ribosome inhibiting antibiotic drugs show an interesting behavior: Although the drug slows down cell growth, it also paradoxically increases the cells concentration of ribosomes. We combine a prior nonlinear model of the energy-biomass balance in undrugged E. coli cells (Maitra and Dill, PNAS 2015) with Michaelis-Menten binding of drugs that inactivate ribosomes. Predictions are in good agreement with experiments on ribosomal concentrations and synthesis rates vs. drug concentrations and growth rates. The model indicates that added drug drives the cell to overproduce ribosomes keeping roughly constant the level of ribosomes producing ribosomal proteins, an important quantity for cell growth. The model also predicts that ribosomal production rates should increase, then decrease with added drug. This model gives insights into cellular driving forces and suggests new experiments.

Biophysics↗

bioRxiv2015

Cell-cell communication enhances the capacity of cell ensembles to sense shallow gradients during morphogenesis

AbstractCollective cell responses to exogenous cues depend on cell-cell interactions. In principle, these can result in enhanced sensitivity to weak and noisy stimuli. However, this has not yet been shown experimentally, and, little is known about how multicellular signal processing modulates single cell sensitivity to extracellular signaling inputs, including those guiding complex changes in the tissue form and function. Here we explored if cell-cell communication can enhance the ability of cell ensembles to sense and respond to weak gradients of chemotactic cues. Using a combination of experiments with mammary epithelial cells and mathematical modeling, we find that multicellular sensing enables detection of and response to shallow Epidermal Growth Factor (EGF) gradients that are undetectable by single cells. However, the advantage of this type of gradient sensing is limited by the noisiness of the signaling relay, necessary to integrate spatially distributed ligand concentration information. We calculate the fundamental sensory limits imposed by this communication noise and combine them with the experimental data to estimate the effective size of multicellular sensory groups involved in gradient sensing. Functional experiments strongly implicated intercellular communication through gap junctions and calcium release from intracellular stores as mediators of collective gradient sensing. The resulting integrative analysis provides a framework for understanding the advantages and limitations of sensory information processing by relays of chemically coupled cells.

Biophysics↗

bioRxiv2015

Exact solutions of coupled multispecies linear reaction-diffusion equations on a uniformly growing domain

Embryonic development involves diffusion and proliferation of cells, as well as diffusion and reaction of molecules, within growing tissues. Mathematical models of these processes often involve reaction-diffusion equations on growing domains that have been primarily studied using approximate numerical solutions. Recently, we have shown how to obtain an exact solution to a single, uncoupled, linear reaction-diffusion equation on a growing domain, 0 < x < L(t), where L(t) is the domain length. The present work is an extension of our previous study, and we illustrate how to solve a system of coupled reaction-diffusion equations on a growing domain. This system of equations can be used to study the spatial and temporal distributions of different generations of cells within a population that diffuses and proliferates within a growing tissue. The exact solution is obtained by applying an uncoupling transformation, and the uncoupled equations are solved separately before applying the inverse uncoupling transformation to give the coupled solution. We present several example calculations to illustrate different types of behaviour. The first example calculation corresponds to a situation where the initially-confined population diffuses sufficiently slowly that it is unable to reach the moving boundary at x = L(t). In contrast, the second example calculation corresponds to a situation where the initially-confined population is able to overcome the domain growth and reach the moving boundary at x = L(t). In its basic format, the uncoupling transformation at first appears to be restricted to deal only with the case where each generation of cells has a distinct proliferation rate. However, we also demonstrate how the uncoupling transformation can be used when each generation has the same proliferation rate by evaluating the exact solutions as an appropriate limit.

Biophysics↗

bioRxiv2015

Tyrosination of α-Tubulin Controls The Initiation of Processive Dynein-Dynactin Motility

Post-translational modifications (PTMs) of /{beta}-tubulin are believed to regulate interactions with microtubule binding proteins. A well-characterized PTM involves the removal and re-ligation of the C-terminal tyrosine on -tubulin, but the purpose of this tyrosination-detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~4-fold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactins microtubule binding p150 subunit rather than dynein itself. Interestingly, on chimeric microtubules, DDB molecules that initiated movement on tyrosinated tubulin continued moving into a region of detyrosinated tubulin. This result indicates that the -tubulin tyrosine facilitates initial motor-tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C-terminal -tubulin tyrosine on dynein-dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein-driven motility in cells.

Biophysics↗

bioRxiv2015

Molecular stripping in the NFκB/IκB/DNA genetic regulatory network

Genetic switches based on the NF{kappa}B/I{kappa}B/DNA system are master regulators of an array of cellular responses. Recent kinetic experiments have shown that I{kappa}B can actively remove NF{kappa}B bound to its genetic sites via a process called \"molecular stripping\". This allows the NF{kappa}B/I{kappa}B/DNA switch to function under kinetic control rather than the thermodynamic control contemplated in the traditional models of gene switches. Using molecular dynamics simulations of coarse grained predictive energy landscape models for the constituent proteins by themselves and interacting with the DNA we explore the functional motions of the transcription factor NF{kappa}B and its various binary and ternary complexes with DNA and the inhibitor I{kappa}B. These studies show that the function of the NF{kappa}B/I{kappa}B/DNA genetic switch is realized via an allosteric mechanism. Molecular stripping occurs through the activation of a domain twist mode by the binding of I{kappa}B which occurs through conformational selection. Free energy calculations for DNA binding show that the binding of I{kappa}B not only results in a significant decrease of the affinity of the transcription factor for the DNA but also kinetically speeds DNA release. Projections of the free energy onto various reaction coordinates reveal the structural details of the stripping pathways.

Biophysics↗

bioRxiv2015

Molecular Simulation of Nonfacilitated Membrane Permeation

This is a review. Non-electrolytic compounds typically cross cell membranes by passive diffusion. The rate of permeation is dependent on the chemical properties of the solute and the composition of the lipid bilayer membrane. Predicting the permeability coefficient of a solute is important in pharmaceutical chemistry and toxicology. Molecular simulation has proven to be a valuable tool for modeling permeation of solutes through a lipid bilayer. In particular, the solubility-diffusion model has allowed for the quantitative calculation of permeability coefficients. The underlying theory and computational methods used to calculate membrane permeability are reviewed. We also discuss applications of these methods to examine the permeability of solutes and the effect of membrane composition on permeability. The application of coarse grain and polarizable models is discussed.

Biophysics↗

bioRxiv2015

Continuum mechanics modeling of Drosophila mesoderm invagination based on spherical harmonics accounts for long-range tissue remodeling

Mechanics plays a key role in the development of higher organisms. However, working towards an understanding of this relationship is complicated by the fact that it has proven difficult to model the link between local forces generated at the subcellular level, and tissue deformation at the whole-embryo level. Here we propose an approach first developed for lipid bilayers and cell membranes, in which force-generation at the cytoskeletal level only enters the shape mechanics calculation in the form of local changes in preferred tissue curvature. This allows us to formulate the continuum mechanics problem purely in terms of tissue strains. Relaxing the system by lowering its mechanical energy yields global morphogenetic predictions that accommodate the tendency towards this local preferred curvature, without explicitly modeling force-generating mechanisms at the molecular level. Our computational framework, which we call SPHARMMECH, extends a three-dimensional spherical harmonics parameterization known as SPHARM to combine this level of abstraction with a sparse shape representation. The integration of these two principles allows computer simulations to be performed in three dimensions, on highly complex shapes, gene expression patterns, and mechanical constraints.\n\nWe demonstrate our approach by modeling mesoderm invagination in the fruit-fly embryo, where local forces generated by the acto-myosin meshwork in the region of the future mesoderm lead to formation of a ventral tissue fold. The process is accompanied by substantial changes in cell shape and long-range cell movements. Applying SPHARM-MECH to whole-embryo live imaging data acquired with light-sheet microscopy reveals significant correlation between calculated and observed tissue movements. Our analysis predicts the observed cell shape anisotropy on the ventral side of the embryo and suggests an active mechanical role of mesoderm invagination in supporting the onset of germ-band extension.

Biophysics↗

bioRxiv2015

On characterizing membrane protein clusters with model-free spatial correlation approaches

Spatial aggregation or clustering of membrane proteins could be important for their functionality, e.g., in signaling, and nanoscale imaging can be used to study its origins, structure and function. Such studies require accurate characterization of clusters, both for absolute quantification and hypothesis testing. A set of model-free quantification approaches -- free of specific cluster models-- have been proposed for this purpose. They include the radius of maximal aggregation ra obtained from the maxima of the empirical Besag L(r) - r function as an estimator of cluster size, and the estimation of various cluster parameters based on an exponential approximation for the Pair Correlation Function(PCF). However, the parameter identifiability and bias and scaling due to their model-free nature are not clear. In practice, the clusters might exhibit specific patterns, and the behavior of these estimators in such cases must be studied. Here, we theoretically analyze these approaches for a set of cluster models, and obtain information about their identifiability and bias. We find that the ratio between ra and true cluster size depends on both the true size as well as the number of clusters per unit area, or other corresponding parameters, in a model-dependent manner. In particular, ra scales with respect to the true size by a factor that can be arbitrarily large, depending on models and parameter values. For the method based on PCF approximation, for most models we analyzed, the ratios between approximate and true model parameters were found to be constants that depend only on models and independent of other parameters. For the models analyzed, this ratio was within {+/-}100%. Our theoretical approach was validated by means of simulations. We also discuss some general issues in inference using second-order spatial properties. While precision could also be key, such information on identifiability and accuracy provides clarity on estimation, can lead to better inference, and can also fuel more accurate method development.

Biophysics↗

bioRxiv2015

Targeted mutations on 3D hub loci alter spatial interaction environment

Many disease-related genotype variations (GVs) reside in non-gene coding regions and the mechanisms of their association with diseases are largely unknown. A possible impact of GVs on disease formation is to alter the spatial organization of chromosome. However, the relationship between GVs and 3D genome structure has not been studied at the chromosome scale. The kilobase resolution of chromosomal structures measured by Hi-C have provided an unprecedented opportunity to tackle this problem. Here we proposed a network-based method to capture global properties of the chromosomal structure. We uncovered that genome organization is scale free and the genomic loci interacting with many other loci in space, termed as hubs, are critical for stabilizing local chromosomal structure. Importantly, we found that cancer-specific GVs target hubs to drastically alter the local chromosomal interactions. These analyses revealed the general principles of 3D genome organization and provided a new direction to pinpoint genotype variations in non-coding regions that are critical for disease formation.

Biophysics↗

bioRxiv2015

Shape Transitions and Chiral Symmetry Breaking in the Energy Landscape of the Mitotic Chromosome

We derive an unbiased information theoretic energy landscape for chromosomes at metaphase using a maximum entropy approach that accurately reproduces the details of the experimentally measured pair-wise contact probabilities between genomic loci. Dynamical simulations using this landscape lead to cylindrical, helically twisted structures reflecting liquid crystalline order. These structures are similar to those arising from a generic ideal homogenized chromosome energy landscape. The helical twist can be either right or left handed so chiral symmetry is broken spontaneously. The ideal chromosome landscape when augmented by interactions like those leading to topologically associating domain (TAD) formation in the interphase chromosome reproduces these behaviors. The phase diagram of this landscape shows the helical fiber order and the cylindrical shape persist at temperatures above the onset of chiral symmetry breaking which is limited by the TAD interaction strength.

Biophysics↗

bioRxiv2015

Modeling Small Non-canonical RNA Motifs with the Rosetta FARFAR Server

Non-canonical RNA motifs help define the vast complexity of RNA structure and function, and in many cases, these loops and junctions are on the order of only 10 nucleotides in size. Unfortunately, despite their small size, there is no reliable method to determine the ensemble of lowest energy structures of junctions and loops at atomic accuracy. This chapter outlines straightforward protocols using a webserver for Rosetta Fragment Assembly of RNA with Full Atom Refinement (FARFAR) (http://rosie.rosettacommons.org/rna_denovo/submit) to model the 3D structure of small non-canonical RNA motifs for use in visualizing motifs and for further refinement or filtering with experimental data such as NMR chemical shifts.

Biophysics↗

bioRxiv2015

Overview: Modeling Heterogeneous Tumor Tissue as a Multiphase Material

Tumors are typically heterogeneous tissues comprised of multiple cell species in addition to extra-cellular matrix (ECM) and water fluid. It is difficult to model these components at the tissue (10-3-10-2m) scale, where individual cells cannot be represented without prohibitive computational burden. Assuming that same-kind components tend to cluster together, a multiphase approach can be applied to represent heterogeneous tumor tissue at this larger physical scale. This method enables simulating mixture of elements within tissues, e.g., geno-/phenotypic heterogeneity underlying mutation- or microenvironment-driven tumor progression. Further, by not explicitly tracking interfaces, this methodology facilitates realistic modeling of tissue in 3-D.

Biophysics↗

bioRxiv2015

Unraveling the mechanism of proton translocation in the extracellular half-channel of bacteriorhodopsin

Bacteriorhodopsin, a light activated protein that creates a proton gradient in halobacteria, has long served as a simple model of proton pumps. Within bacteriorhodopsin, several key sites undergo protonation changes during the photocycle, moving protons from the higher pH cytoplasm to the lower pH extracellular side. The mechanism underlying the long-range proton translocation between the central (the retinal Schiff base SB216, D85 and D212) and exit clusters (E194 and E204) remains elusive. To obtain a dynamic view of the key factors controlling proton translocation, a systematic study using molecular dynamics simulation was performed for eight bacteriorhodopsin models varying in retinal isomer and protonation of the SB216, D85, D212 and E204. The side-chain orientation of R82 is determined primarily by the protonation states of the residues in the EC. The side-chain reorientation of R82 modulates the hydrogen-bond network and consequently possible pathways of proton transfer. Quantum mechanical intrinsic reaction coordinate calculations of proton-transfer in the methyl guanidinium-hydronium-hydroxide model system show that proton transfer via a guanidinium group requires an initial geometry permitting proton donation and acceptance by the same amine. In all the models, R82 can form proton wires with both the CC and the EC connected by the same amine. Alternatively, rare proton wires for proton transfer from the CC to the EC without involving R82 were found in an O state where the proton on D85 is transferred to D212.

Biophysics↗

bioRxiv2015

The genomic basis of electrotaxis in Dictyostelium discoideum: Electric field sensitive amino acids are dynamically encoded en masse for the streaming-stage proteome

Electrotaxis plays a critical role in developmental cell migration, axon growth cone guidance, epithelial wound healing, tissue regeneration, and the degree of invasiveness characterizing different cancer cell lines. During electrotaxis in a direct current electric field (EF), a cell migrates preferentially either towards the anode or cathode depending on the cell-type. However, the types and ranges of mechanisms coupling trans-cellular electric fields to cellular EF-sensitive signaling systems are largely unknown. To address this cell biological phenomenon, I use transcriptomic data from a developmental genetic model in which multicellular social aggregation is induced by starvation of amoeboid cells. I find that the developmental proteome expressed during the streaming aggregation stage is measurably and substantially enriched in charged and highly polar amino acids relative to the proteomes of either the unicellular amoeboid or the multicellular fruiting body. This large-scale coding augmentation of EF-sensitive amino acid residues in thousands of streaming-specific proteins is accompanied by a proportional coding decrease in the number of small, uncharged amino acid residues. I also confirm an expected coding increase of biosynthetically costly amino acids in the proteome of the satiated feeding-stage amoeboid. These findings suggest that electrotactic capability is encoded broadly in the genetically regulated deployment of a developmental proteome with augmented EF-sensitivity. These results signify that extreme, nonuniform, evolutionary constraints can be exerted on the amino acid composition of an organisms proteome.

Biophysics↗

bioRxiv2016

Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves

Despite the central role that antibodies play in the adaptive immune system and in biotechnology, much remains unknown about the quantitative relationship between an antibodys amino acid sequence and its antigen binding affinity. Here we describe a new experimental approach, called TiteSeq, that is capable of measuring binding titration curves and corresponding affinities for thousands of variant antibodies in parallel. The measurement of titration curves eliminates the confounding effects of antibody expression and stability that arise in standard deep mutational scanning assays. We demonstrate Tite-Seq on the CDR1H and CDR3H regions of a well-studied scFv antibody. Our data shed light on the structural basis for antigen binding affinity and suggests a role for secondary CDR loops in establishing antibody stability. Tite-Seq fills a large gap in the ability to measure critical aspects of the adaptive immune system, and can be readily used for studying sequence-affinity landscapes in other protein systems.

Biophysics↗