SEARCH · Search bioRxiv

Results for “Biophysics”

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources

bioRxiv2016

Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules

We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2 m holes of the support films. Randomly biotinylated 70 S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.

Biophysics↗

bioRxiv2016

Experiment-friendly kinetic analysis of single molecule data in and out of equilibrium

We present a simple and robust technique to extract kinetic rate models and thermodynamic quantities from single molecule time traces. SMACKS (Single Molecule Analysis of Complex Kinetic Sequences) is a maximum likelihood approach that works equally well for long trajectories as for a set of short ones. It resolves all statistically relevant rates and also their uncertainties. This is achieved by optimizing one global kinetic model based on the complete dataset, while allowing for experimental variations between individual trajectories. In particular, neither a priori models nor equilibrium have to be assumed. The power of SMACKS is demonstrated on the kinetics of the multi-domain protein Hsp90 measured by smFRET (single molecule Forster resonance energy transfer). Experiments in and out of equilibrium are analyzed and compared to simulations, shedding new light on the role of Hsp90s ATPase function. SMACKS pushes the boundaries of single molecule kinetics far beyond current methods.

Biophysics↗

bioRxiv2016

Stochastic models of gene transcription with upstream drives: exact solution and sample path characterization

Gene transcription is a highly stochastic and dynamic process. As a result, the mRNA copy number of a given gene is heterogeneous both between cells and across time. We present a framework to model gene transcription in populations of cells with time-varying (stochastic or deterministic) transcription and degradation rates. Such rates can be understood as upstream cellular drives representing the effect of different aspects of the cellular environment. We show that the full solution of the master equation contains two components: a model-specific, upstream effective drive, which encapsulates the effect of the cellular drives (e.g., entrainment, periodicity or promoter randomness), and a downstream transcriptional Poissonian part, which is common to all models. Our analytical framework allows us to treat cell-to-cell and dynamic variability consistently, unifying several approaches in the literature. We apply the obtained solution to characterize several gene transcription models of experimental relevance, and to explain the influence on gene transcription of synchrony, stationarity, ergodicity, as well as the effect of time-scales and other dynamic characteristics of drives. We also show how the solution can be applied to the analysis of single-cell data, and to reduce the computational cost of sampling solutions via stochastic simulation.

Biophysics↗

bioRxiv2016

Rapid Dynamics of Cell-Shape Recovery in Response to Local Deformations

It is vital that cells respond rapidly to mechanical cues within their microenvironment through changes in cell shape and volume, which rely upon the mechanical properties of cells highly interconnected cytoskeletal networks and intracellular fluid redistributions. While previous research has largely investigated deformation mechanics, we now focus on the immediate cell-shape recovery response following mechanical perturbation by inducing large, local, and reproducible cellular deformations using AFM. By continuous imaging within the plane of deformation, we characterize the membrane and cortical response of HeLa cells to unloading, and model the recovery via overdamped viscoelastic dynamics. Importantly, the majority (90%) of HeLa cells recover their cell shape in < 1s. Despite actin remodelling on this time scale, we show that cell recovery time is not affected by load duration nor magnitude. To further explore this rapid recovery response, we expose cells to cytoskeletal destabilizers and osmotic shock conditions, which exposes the interplay between actin and osmotic pressure. We show that the rapid dynamics of recovery depend crucially on intracellular pressure, and provide strong evidence that cortical actin is the key regulator in the cell-shape recovery processes, in both cancerous and non-cancerous epithelial cells.

Biophysics↗

bioRxiv2016

Controlling noise in the timing of intracellular events: A first-passage time approach

In the noisy cellular environment, gene products are subject to inherent random fluctuations in copy numbers over time. How cells ensure precision in the timing of key intracellular events, in spite of such stochasticity is an intriguing fundamental problem. We formulate event timing as a first-passage time problem, where an event is triggered when the level of a protein crosses a critical threshold for the first time. Novel analytical calculations are preformed for the first-passage time distribution in stochastic models of gene expression, including models with feedback regulation. Derivation of these formulas motivates an interesting question: is there an optimal feedback strategy to regulate the synthesis of a protein to ensure that an event will occur at a precise time, while minimizing deviations or noise about the mean. Counter-intuitively, results show that for a stable long-lived protein, the optimal strategy is to express the protein at a constant rate without any feedback regulation, and any form of feedback (positive, negative or any combination of them) will always amplify noise in event timing. In contrast, a positive feedback mechanism provides the highest precision in timing for an unstable protein. These theoretical results explain recent experimental observations of single-cell lysis times in bacteriophage{lambda} . Here, lysis of an infected bacterial cell is orchestrated by the expression and accumulation of a stable{lambda} protein up to a threshold, and precision in timing is achieved via feedforward, rather than feedback control. Our results have broad implications for diverse cellular processes that rely on precise temporal triggering of events.

Biophysics↗

bioRxiv2016

Stabilizing membrane domains antagonizes n-alcohol anesthesia

Diverse molecules induce general anesthesia with potency strongly correlated both with their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma membrane derived vesicles by lowering the critical temperature (Tc) for phase separation. Here we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on Tc. First we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described intoxication reversers raise Tc and counter ethanols effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that hydrostatic pressure, long known to reverse anesthesia, also raises Tc in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together,these results demonstrate that {Delta}Tc predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.

Biophysics↗

bioRxiv2016

Effect of X-ray irradiation on ancient DNA in sub-fossil bones - Guidelines for safe x-ray imaging.

Sub-fossilised remains may still contain highly degraded ancient DNA (aDNA) useful for palaeogenetic investigations. Whether X-ray computed [micro-] tomography ([]CT) imaging of these fossils may further damage aDNA remains debated. Although the effect of X-ray on DNA in living organisms is well documented, its impact on aDNA molecules is unexplored.\n\nHere we investigate the effects of synchrotron X-ray irradiation on aDNA from Pleistocene bones. A clear correlation appears between decreasing aDNA quantities and accumulating X-ray dose-levels above 2000 Gray (Gy). We further find that strong X-ray irradiation reduces the amount of nucleotide misincorporations at the aDNA molecule ends. No representative effect can be detected for doses below 200 Gy. Dosimetry shows that conventional CT usually does not reach the risky dose level, while classical synchrotron imaging can degrade aDNA significantly. Optimised synchrotron protocols and simple rules introduced here are sufficient to ensure that fossils can be scanned without impairing future aDNA studies.

Biophysics↗

bioRxiv2016

Actomyosin Dynamics Determine the Extension and Retraction of Filopodia on Neuronal Dendrites

Impact StatementIn this study, using a combination of computational and experimental approaches we show that a complex dynamic behavior of dendritic filopodia that is essential for synaptogenesis is explained by an interplay among forces generated by actin retrograde flow, myosin contractility, and substrate adhesion.\n\nAbstractDendritic filopodia are actin-filled dynamic subcellular structures that sprout on neuronal dendrites during neurogenesis. The exploratory motion of the filopodia is crucial for synaptogenesis but the underlying mechanisms are poorly understood. To study the filopodial motility, we collected and analyzed image data on filopodia in cultured rat hippocampal neurons. We hypothesized that mechanical feedback among the actin retrograde flow, myosin activity and substrate adhesion gives rise to various filopodial behaviors. We have formulated a minimal one-dimensional partial differential equation model that reproduced the range of observed motility. To validate our model, we systematically manipulated experimental correlates of parameters in the model: substrate adhesion strength, actin polymerization rate, myosin contractility and the integrity of the putative microtubule-based barrier at the filopodium base. The model predicts the response of the system to each of these experimental perturbations, supporting the hypothesis that our actomyosin-driven mechanism controls dendritic filopodia dynamics.

Biophysics↗

bioRxiv2016

The mitotic kinesin-14 KlpA contains a context-dependent directionality switch

Kinesins are microtubule-based motor proteins that convert chemical energy from ATP hydrolysis into mechanical work for a variety of essential intracellular processes. Kinesin-14s (i.e. kinesins with a C-terminal motor domain) are commonly considered to be nonprocessive minus end-directed motors that mainly function for mitotic spindle assembly and maintenance. Here, we show that KlpA - a mitotic kinesin-14 motor from the filamentous fungus Aspergillus nidulans - contains a context-dependent directionality switch. KlpA exhibits canonical minus end-directed motility inside microtubule bundles, but on individual microtubules it unexpectedly moves processively toward the plus ends. Removal of the N-terminal nonmotor microtubule-binding domain renders KlpA diffusive on individual microtubules but does not abolish its minus end-directed motility to collectively glide microtubules, suggesting that the nonmotor microtubule-binding domain likely acts as a switch for controlling the direction of KlpA motility. Collectively, these findings provide important insights into the mechanism and regulation of KlpA functions inside the mitotic spindle.

Biophysics↗

bioRxiv2016

Dynamic Nucleosome Movement Provides Structural Information of Topological Chromatin Domains in Living Human Cells

The mammalian genome is organized into submegabase-sized chromatin domains (CDs) including topologically associating domains, which have been identified using chromosome conformation capture-based methods. Single-nucleosome imaging in living mammalian cells has revealed subdiffusively dynamic nucleosome movement. It is unclear how single nucleosomes within CDs fluctuate and how the CD structure reflects the nucleosome movement. Here, we present a polymer model wherein CDs are characterized by fractal dimensions and the nucleosome fibers fluctuate in a viscoelastic medium with memory. We analytically show that the mean-squared displacement (MSD) of nucleosome fluctuations within CDs is subdiffusive. The diffusion coefficient and the subdiffusive exponent depend on the structural information of CDs. This analytical result enabled us to extract information from the single-nucleosome imaging data for HeLa cells. Our observation that the MSD is lower at the nuclear periphery region than the interior region indicates that CDs in the heterochromatin-rich nuclear periphery region are more compact than those in the euchromatin-rich interior region with respect to the fractal dimensions as well as the size. Finally, we evaluated that the average size of CDs is in the range of 100-500 nm and that the relaxation time of nucleosome movement within CDs is a few seconds. Our results provide physical and dynamic insights into the genome architecture in living cells.\n\nAuthor SummaryThe mammalian genome is partitioned into topological chromatin domains (CDs) in the living cell nuclei. Gene expression is highly regulated within CDs according to their structure, whereas chromatin itself is highly dynamic. This raises the following question: how is the CD structure in such dynamic chromatin? We developed a conceptual framework that unifies chromatin dynamics and structure. Using a polymer model with a fractal domain structure in a viscoelastic medium, we analytically show that nucleosome movement is subdiffusive and depends on CD structure. Hence, structural information can be extracted based on nucleosome movement in living cells with single-particle tracking experiments. This framework provides physical insights into the relationship between dynamic genome organization and gene expression.

Biophysics↗

bioRxiv2016

Probing cytoplasmic viscosity in the confined geometry of tip-growing plant cells via FRAP

Fluorescence Recovery After Photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus or cell membrane. While many FRAP models have been developed over the past decades, the influence of the complex boundaries of three-dimensional cellular geometries on the recovery curves, in conjunction with ROI and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied. Here, we developed a three-dimensional computational model of the FRAP process that incorporates particle diffusion, cell boundary effects, and the optical properties of the scanning confocal microscope, and validated this model using the tip-growing cells of Physcomitrella patens. We then show how these cell boundary and optical effects confound the interpretation of FRAP recovery curves, including the number of dynamic states of a given fluorescent protein, in a wide range of cellular geometries-both in two and three dimensions-namely nuclei, filopodia, and lamellipodia of mammalian cells, and in cell types such as the budding yeast, S. pombe, and tip-growing plant cells. We explored the performance of existing analytical and algorithmic FRAP models in these various cellular geometries, and determined that the VCell VirtualFRAP tool provides the best accuracy to measure diffusion coefficients. Our computational model is not limited only to these cells types, but can easily be extended to other cellular geometries via the graphical Java-based application we also provide. This particle-based simulation-called the Digital Confocal Microscopy Suite, DCMS-can also perform fluorescence dynamics assays, such as Number and Brightness (N&B), Fluorescence Correlation Spectroscopy (FCS), Raster Image Correlation Spectroscopy (RICS), and could help shape the way these techniques are interpreted.

Biophysics↗

bioRxiv2016

Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2

By reaching near-atomic resolution for a wide range of specimens, single-particle cryo-EM structure determination is transforming structural biology. However, the necessary calculations come at increased computational costs, introducing a bottleneck that is currently limiting throughput and the development of new methods. Here, we present an implementation of the O_SCPCAPRELIONC_SCPCAP image processing software that uses graphics processors (GPUs) to address the most computationally intensive steps of its cryo-EM structure determination workflow. Both image classification and high-resolution refinement have been accelerated up to 40-fold, and template-based particle selection has been accelerated almost 1000-fold on desktop hardware. Memory requirements on GPUs have been reduced to fit widely available hard-ware, and we show that the use of single precision arithmetic does not adversely affect results. This enables high-resolution cryo-EM structure determination in a matter of days on a single workstation.

Biophysics↗

bioRxiv2016

A wireless centrifuge force microscope (CFM) enables multiplexed single-molecule experiments in a commercial centrifuge

The centrifuge force microscope (CFM) was recently introduced as a platform for massively parallel single-molecule manipulation and analysis. Here we developed a low-cost and selfcontained CFM module that works directly within a commercial centrifuge, greatly improving accessibility and ease of use. Our instrument incorporates research grade video microscopy, a power source, a computer, and wireless transmission capability to simultaneously monitor many individually tethered microspheres. We validated the instrument by performing single-molecule force shearing of short DNA duplexes. For a 7 bp duplex, we collected over 1000 statistics of force dependent shearing kinetics from 2 pN to 12 pN with dissociation times in the range of 10-100 seconds. We extended the measurement to a 10 bp duplex, applying a 12 pN force clamp and directly observing single-molecule dissociation over an 85 minute experiment. Our new CFM module facilitates simple and inexpensive experiments that dramatically improve access to singlemolecule analysis.

Biophysics↗

bioRxiv2016

A NOVEL METHOD FOR MEASURING TEXTURE USING A FOOD PROCESSOR

Measurement of food texture is becoming increasingly important to help prevent mis-swallowing in elderly individuals. However, it is difficult to estimate food texture for mastication and swallowing. In this study, a mixing-recording method was applied to measure food texture. A commercial food processor was used to homogenize food gels, and the torque was monitored as an electric current value, which was correlated with the viscosity of Newtonian fluid. Agar and gelatin gels at several concentrations were applied to the mixing-recording system, and individual mixing curves were obtained. The current values had a different tendency at each mixing stage. At the beginning of the mixing, when lumps of the original gel were present, current values of gelatin were higher than those of agar. From the middle of mixing, the fractured pastes of high-concentration agar gels had little mobility, and current values decreased, indicating that gelatin was more cohesive than agar. The current values of low-concentration agar and gelatin were correlated with the hardness of the gels and were aligned on the same correlation curve, indicating that the viscosity of the fractured paste was correlated with the original gel hardness under certain conditions. Moreover, measurements of agar gels of different sizes revealed that the texture was size dependent. Thus, the mixing-recording method had several advantages over conventional methods, including time- and size-dependent fracturing and bolus properties, and could be useful for the measurement of food texture in terms of mastication and swallowing.

Biophysics↗

bioRxiv2016

FF12MC: A revised AMBER forcefield and new protein simulation protocol

Specialized to simulate proteins in molecular dynamics (MD) simulations with explicit solvation, FF12MC is a combination of a new protein simulation protocol employing uniformly reduced atomic masses by tenfold and a revised AMBER forcefield FF99 with (i) shortened CH bonds, (ii) removal of torsions involving a nonperipheral sp3 atom, and (iii) reduced 1-4 interaction scaling factors of torsions{phi} and{psi} This article reports that in multiple, distinct, independent, unrestricted, unbiased, isobaric-isothermal, and classical MD simulations FF12MC can (i) simulate the experimentally observed flipping between left-and right-handed configurations for C14-C38 of BPTI in solution, (ii) autonomously fold chignolin, CLN025, and Trp-cage with folding times that agree with the experimental values, (iii) simulate subsequent unfolding and refolding of these miniproteins, and (iv) achieve a robust Z score of 1.33 for refining protein models TMR01, TMR04, and TMR07. By comparison, the latest general-purpose AMBER forcefield FF14SB locks the C14-C38 bond to the right-handed configuration in solution under the same protein simulation conditions. Statistical survival analysis shows that FF12MC folds chignolin and CLN025 in isobaric-isothermal MD simulations 2-4 times faster than FF14SB under the same protein simulation conditions. These results suggest that FF12MC may be used for protein simulations to study kinetics and thermodynamics of miniprotein folding as well as protein structure and dynamics.

Biophysics↗

bioRxiv2016

Anisotropic Correction of Beam-induced Motion for Improved Single-particle Electron Cryo-microscopy

Correction of electron beam-induced sample motion is one of the major factors contributing to the recent resolution breakthroughs in cryo-electron microscopy. Improving the accuracy and efficiency of motion correction can lead to further resolution improvement. Based on observations that the electron beam induces doming of the thin vitreous ice layer, we developed an algorithm to correct anisotropic image motion at the single pixel level across the whole frame, suitable for both single particle and tomographic images. Iterative, patch-based motion detection is combined with spatial and temporal constraints and dose weighting. The multi-GPU accelerated program, MotionCor2, is sufficiently fast to keep up with automated data collection. The result is an exceptionally robust strategy that can work on a wide range of data sets, including those very close to focus or with very short integration times, obviating the need for particle polishing. Application significantly improves Thon ring quality and 3D reconstruction resolution.

Biophysics↗

bioRxiv2016

An internal disulfide locks a misfolded aggregation-prone intermediate in cataract-linked mutants of human γD-crystallin

Considerable mechanistic insight has been gained into amyloid aggregation; however, a large class of non-amyloid protein aggregates are considered \"amorphous,\" and in most cases little is known about their mechanisms. Amorphous aggregation of {gamma}-crystallins in the eye lens causes a widespread disease of aging, cataract. We combined simulations and experiments to study the mechanism of aggregation of two {gamma}D-crystallin mutants, W42R and W42Q - the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys32 and Cys41, was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro. Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal {beta}-hairpin and rearrangement of the native {beta}-sheet topology. Specific binding between the extruded hairpin and a distal {beta}-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation.

Biophysics↗

bioRxiv2016

Lateral pressure profile in bent lipid membranes and mechanosensitive channel gating

This review describes the analytical calculation of lateral pressure profile in the hydrophobic part of the lipid bilayer with finite curvature based on previously developed microscopic model for lipid hydrocarbon chains. According to this theory the energy per unit chain is represented as energy of flexible string (Eulers elastic beam of finite thickness) and interaction between chains is considered as an entropic repulsion. This microscopic theory allows to obtain expression for lateral pressure distribution in bent bilayer if treating a bending as a small deviation from the flat membrane conformation and using perturbation theory. Because lateral pressure distribution is related to elastic properties of lipid bilayer then the first moment of lateral pressure and the expression for bending modulus may be derived from this theoretical model. Finally one can estimate the energy difference between two various conformational states of mechanosensitive channel embedded into the bilayer with pressure profile [Pcy]t(z).

Biophysics↗