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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 685 records · Page 38Linked to original sources

bioRxiv2017

Chemotaxis microsimulation: On the gain in nutrient uptake and bacterial cell division with chemotaxis mechanism

Bacterial swimming alternates between straight runs for several seconds and tumbles into random directions. Chemotactic bacteria remember nutrient sensing history, change tumble frequency to move toward nutrients. A question that has not been addressed is the significance of the nutrition gain and multiplication of bacterial population with chemotaxis mechanism. To quantify these effects, we introduce a microsimulation model, which seamlessly integrates detailed observations and assumptions about single bacterial tumbles, noisy sensing and nutrient uptake for studying up to a few millions of them in a population. We use the model to simulate absorption of nutrients from lysis and agar plates. Contrary to an intuitive feeling that chemotaxis could be useful under nutrient starvation, we see a significant effect only under nutrient rich conditions where bacteria with chemotaxis outgrow their non-chemotactic counterparts by hundreds of times. The model offers the flexibility to study the consequences of newer assumptions, and experimental conditions.\n\nAuthor SummaryChemotaxis is a mechanism that helps bacteria navigate towards nutrients. Several aspects of the mechanism have been well studied over the past 50 years. As most bacterial mechanisms are helpful evolutionarily to survive and to multiply, it would be a natural question to ask how much this swim helps bacteria to gain nutrition and consequently to multiply. However to our knowledge this question has not been asked. We develop a model that integrates bacterial motion, with sensing and nutrient uptake and show that only under nutrient rich conditions this mechanism helps.

biophysics↗

bioRxiv2017

Elucidating interplay of speed and accuracy in biological error correction

One of the most fascinating features of biological systems is the ability to sustain high accuracy of all major cellular processes despite the stochastic nature of underlying chemical processes. It is widely believed that such low errors are the result of the error correcting mechanism known as kinetic proofreading. However, it is usually argued that enhancing the accuracy should result in slowing down the process leading to so-called speed-accuracy trade-off. We developed a discrete-state stochastic framework that allowed us to investigate the mechanisms of the proofreading using the method of first-passage processes. With this framework, we simultaneously analyzed speed and accuracy of the two fundamental biological processes, DNA replication and tRNA selection during the translation. The results indicate that speed-accuracy trade-off is not always observed. However, when the trade-off is present, the biological systems tend to optimize the speed rather than the accuracy of the processes, as long as the error level is tolerable. Additional constraints due to the energetic cost of proofreading also play a role in the error correcting process. Our theoretical findings provide a new microscopic picture of how complex biological processes are able to function so fast with a high accuracy.

biophysics↗

bioRxiv2017

Astral microtubule dynamics regulate anaphase * oscillation onset and set a robust final position * of the C. elegans zygote spindle.

During the asymmetric division of the Caenorhabditis elegans nematode zygote, the polarity cues distribution and daughter cell fates depend on the correct positioning of the mitotic spindle, which results from both centering and cortical pulling forces. Revealed by anaphase spindle rocking, these pulling forces are regulated by the force generator dynamics, which are in turn consequent of mitotic progression. We found a novel, additional, regulation of these forces by the spindle position. It controls astral microtubule availability at the cortex, on which the active force generators can pull. Importantly, this positional control relies on the polarity dependent LET-99 cortical band, which restricts or concentrates generators to a posterior crescent. After delaying anaphase onset, we detected this positional pulling force regulation in C. elegans as a precocious spindle rocking with respect to anaphase onset. We ascribed this control to the microtubule dynamics at the cortex. Indeed, in mapping the cortical contacts, we found a correlation between the centrosome-cortex distance and the microtubule contact density. In turn, it modulates pulling force generator activity. We modelled this control, predicting and experimentally validating that the posterior crescent extent controlled where the anaphase oscillations started, in addition to mitotic progression. We found in particular that the oscillation onset position resists changes in cellular geometry and moderate variations of active force generator count. Finally, we propose that spatially restricting force generator to a posterior crescent sets the spindles final position, reflecting polarity through the LET-99 dependent restriction of force generators to a posterior crescent. This regulation superimposes that of force generator processivity. This novel control confers a low dependence on microtubule and active force generator exact numbers or dynamics, provided that they exceed the threshold needed for posterior displacement. Interestingly, this robustness originates in cell mechanics rather than biochemical networks.

biophysics↗

bioRxiv2017

Asymmetric adhesion of rod-shaped bacteria controls microcolony morphogenesis

Bacterial biofilms are spatially structured communities, within which bacteria can differentiate depending on environmental conditions. During biofilm formation, bacteria attach to a surface and use cell-cell contacts to convey the signals required for the coordination of biofilm morphogenesis. How bacteria can maintain both substrate adhesions and cell-cell contacts during the expansion of a microcolony is still a critical yet poorly understood phenomenon. Here, we describe the development of time-resolved methods to measure substrate adhesion at the single cell level during the formation of E. coli and P. aeruginosa microcolonies. We show that bacterial adhesion is asymmetrically distributed along the cell body. Higher adhesion forces at old poles put the daughter cells under tension and force them to slide along each other. These rearrangements increase cell-cell contacts and the circularity of the colony. We propose a mechanical model based on the microscopic details of adhesive links, which recapitulates microcolony morphogenesis and quantitatively predicts bacterial adhesion from simple time lapse movies. These results explain how the distribution of adhesion forces at the subcellular level directs the shape of bacterial colonies, which ultimately dictates the circulation of secreted signals.

biophysics↗

bioRxiv2017

Explicit Modeling of RNA Stability Improves Large-Scale Inference of Transcription Regulation

Inference of eukaryotic transcription regulatory networks remains challenging due to the large number of regu-lators, combinatorial interactions, and redundant pathways. Even in the model system Saccharomyces cerevisiae, inference has performed poorly. Most existing inference algorithms ignore crucial regulatory components, like RNA stability and post-transcriptional modulation of regulators. Here we demonstrate that explicitly modeling tran-scription factor activity and RNA half-lives during inference of a genome-wide transcription regulatory network in yeast not only advances prediction performance, but also produces new insights into gene-and condition-specific variation of RNA stability. We curated a high quality gold standard reference network that we use for priors on network structure and model validation. We incorporate variation of RNA half-lives into the Inferelator inference framework, and show improved performance over previously described algorithms and over implementations of the algorithm that do not model RNA degradation. We recapitulate known condition-and gene-specific trends in RNA half-lives, and make new predictions about RNA half-lives that are confirmed by experimental data.

biophysics↗

bioRxiv2017

Compaction of Duplex Nucleic Acids upon Native Electrospray Mass Spectrometry

Native mass spectrometry coupled to ion mobility spectrometry is a promising tool for structural biology. Intact complexes can be transferred to the mass spectrometer and, if native conformations survive, collision cross sections give precious information on the structure of each species in solution. Based on several successful reports for proteins and their complexes, the conformation survival becomes more and more taken for granted. Here we report on the fate of nucleic acids conformation in the gas phase. Disturbingly, we found that DNA and RNA duplexes, at the electrospray charge states naturally obtained from native solution conditions ([≥] 100 mM aqueous NH4OAc), are significantly more compact in the gas phase compared to the canonical solution structures. The compaction is observed for short (12-bp) and long (36-bp) duplexes, and for DNA and RNA alike. Molecular modeling (density functional calculations on small helices, semi-empirical calculations on up to 12-bp, and molecular dynamics on up to 36-bp duplexes) demonstrates that the compaction is due to phosphate group self-solvation prevailing over Coulomb-driven expansion. Molecular dynamics simulations starting from solution structures do not reproduce the experimental compaction. To be experimentally relevant, molecular dynamics sampling should reflect the progressive structural rearrangements occurring during desolvation. For nucleic acid duplexes, the compaction observed for low charge states results from novel phosphate-phosphate hydrogen bonds formed across both grooves at the very late stages of electrospray.

biophysics↗

bioRxiv2017

Focus: The interface between data collection and data processing in cryo-EM

We present a new software package called Focus that interfaces cryo-transmission electron microscopy (cryo-EM) data collection with computer image processing. Focus creates a user-friendly environment to import and manage data recorded by direct electron detectors and perform elemental image processing tasks in a high-throughput manner while new data is being acquired at the microscope. It provides the functionality required to remotely monitor the progress of data collection and data processing, which is essential now that automation in cryo-EM allows a steady flow of images of single particles, two-dimensional crystals, or electron tomography data to be recorded in overnight sessions. The rapid detection of any errors that may occur greatly increases the productivity of recording sessions at the electron microscope.

biophysics↗

bioRxiv2017

Lipid order degradation in autoimmune demyelination probed by polarization resolved coherent Raman microscopy

Myelin around axons is currently widely studied by structural analyses and large scale imaging techniques, with the goal to decipher its critical role in neuronal protection. While there is strong evidence that in myelin, lipid composition and lipid membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible with in vivo observations. In this work, we study and quantify molecular order of lipids in myelin at sub-diffraction scales, using label-free polarization resolved Coherent Anti Stokes Raman (PR-CARS), which exploits CARS sensitivity to coupling between light polarization and oriented molecular vibrational bonds. Importantly, the method does not use any a priori parameters in the sample such as lipid type, orientational organization and composition. We show that lipid molecular order of myelin in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits to unravel molecular-scale perturbations of lipid layers at early stage of the demyelination progression, while the membrane architecture at the mesoscopic scale (here about 100 nm) seems much less affected. Such information cannot be brought by pure morphological observation and opens new prospectives towards molecular-scale understanding of neurodegenerative diseases.

biophysics↗

bioRxiv2017

The Rosetta all-atom energy function for macromolecular modeling and design

Over the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to design of nanomaterials, protein therapeutics, and vaccines. Central to Rosettas success is the energy function: amodel parameterized from small molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta energy function, beta_nov15. Applying these concepts,we explain how to use Rosetta energies to identify and analyze the features of biomolecular models.Finally, we discuss the latest advances in the energy function that extend capabilities from soluble proteins to also include membrane proteins, peptides containing non-canonical amino acids, carbohydrates, nucleic acids, and other macromolecules.

biophysics↗

bioRxiv2017

Deconvolution of membrane currents for low-crosstalk high-resolution membrane capacitance measurements

Many cellular activities involving cell membrane manifest through changes in membrane capacitance. The accuracy and precision of cell membrane capacitance measurement is limited by the measuring system, which distorts membrane current responses used for analysis of impedance parameters. We developed a practical deconvolution procedure for reconstruction of the time course of membrane current and applied it to current responses recorded at step voltage changes by the whole-cell patch-clamp technique. In contrast to the recorded current responses, the reconstructed current responses could be exactly described by adequate impedance models of the recorded circuit. Deconvolution of membrane currents improved the performance of the square-wave method of high-resolution membrane capacitance recording by providing higher accuracy in a wider range of cell sizes and by eliminating cross-talk errors in parameter estimates. This allowed resolving instabilities in the recording conditions arising from parasitic capacitance and seal resistance variation. Complex tests on hardware models, on simulated data sets, and on living cells confirmed reliability of the deconvolution method for membrane capacitance recording. The aptitude of the method was demonstrated in isolated cardiac myocytes by recording spontaneous vesicular events, by discerning formation of a fusion pore, and by revealing artefacts due to unstable seal resistance.

biophysics↗

bioRxiv2017

Small teams of myosin Vc coordinate their stepping for efficient cargo transport on actin bundles

Myosin Vc (myoVc) is unique among vertebrate class V myosin isoforms in that it requires teams of motors to transport cargo. Single molecules of myoVc cannot take multiple steps on single actin filaments, in stark contrast to the well-studied myosin Va (myoVa) isoform. Consistent with in vivo studies (1), only teams of myoVc motors can move continuously on actin bundles at physiologic ionic strength (2), raising the question of how motor-motor interactions cause this preference. Here, using DNA nanostructures as synthetic cargos for linking defined numbers of myoVa or myoVc molecules, we compared the stepping behavior of myoVa versus myoVc teams, and myoVc stepping patterns on single actin filaments versus actin bundles. Run lengths of both myoVa and myoVc teams increased with motor number, but the run lengths of myoVc teams were longer on actin bundles than on filaments. By resolving the stepping behavior of individual myoVc motors with a Qdot bound to the motor domain, we found that coupling of two myoVc molecules significantly decreases futile back/side steps, which were frequently observed for single myoVc motors. Data showing how changes in the inter-motor distance of two coupled myoVc motors affected stepping dynamics suggested that mechanical tension coordinates the stepping behavior of two molecules for efficient directional motion. Our study thus provides a molecular basis to explain how teams of myoVc motors are suited to transport cargos such as zymogen granules on actin bundles.

biophysics↗

bioRxiv2017

Free energy calculations suggest a mechanism for Na+/K+-ATPase ion selectivity

Na+/K+-ATPase transports Na+ and K+ ions across the cell membrane via an ion binding site made alternatively accessible to the intra- and extracellular milieu by conformational transitions that confer marked changes in ion binding stoichiometry and selectivity. To probe the mechanism of these changes, we used molecular simulation approaches to identify the protonation state of Na+ and K+ coordinating residues in E1P and E2P conformations. Further analysis of these simulations revealed a novel molecular mechanism responsible for the change in protonation state: the conformation-dependent binding of an anion (a chloride ion in our simulations) to a previously unrecognized cytoplasmic site in the loop between transmembrane helices 8 and 9, which influences the electrostatic potential of the crucial Na+-coordinating residue D926. This mechanistic model is consistent with experimental observations and provides a molecular-level picture of how E1P to E2P enzyme conformational transitions are coupled to changes in ion binding stoichiometry and selectivity.

biophysics↗

bioRxiv2017

Polarity sensitive probes for super resolution STED microscopy

The lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed (or ordered). Polarity sensitive dyes are powerful tools to characterize such lipid membrane order, employing for example confocal and two-photon microscopy. The investigation of potential lipid nanodomains, however, requires the use of super resolution microscopy. Here, we test the performance of the polarity sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA and NR12S in super resolution STED microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles show the high potential of these dyes for probing nanoscale membrane heterogeneity.

biophysics↗

bioRxiv2017

Exploring the Complex Dynamics of an Ion Channel Voltage Sensor Domain via Computation

Voltage-gated ion channels are ubiquitous proteins that orchestrate electrical signaling across excitable membranes. Key to their function is activation of the voltage sensor domain (VSD), a transmembrane four alpha-helix bundle that triggers channel opening. Modeling of currents from electrophysiology experiments yields a set of kinetic parameters for a given channel, but no direct molecular insight. Here we use molecular dynamics (MD) simulations to determine the free energy landscape of VSD activation and to, ultimately, predict the time evolution of the resulting gating currents. Our study provides the long-sought-for bridge between electrophysiology and microscopic molecular dynamics and confirms, as already suggested on the basis of experiments, that rate-limiting barriers play a critical role in activation kinetics.

biophysics↗

bioRxiv2017

Analysis of the Quality of Macromolecular Structures

Structure determination utilizing X-ray crystallography involves collection of diffraction data, determination of initial phases followed by iterative rounds of model building and crystallographic refinement to improve the phases and minimize the differences between calculated and observed structure factors. At each of these stages, a variety of statistical filters exist to ensure appropriate validation. Biologically important observations often come from interpretations of signals that need to be carefully deciphered from noise and therefore human intervention is as important as the automated filters. Currently, all structural data are deposited in the Protein Data Bank and this repository is continuously evolving to incorporate possible new improvements in macromolecular crystallography. The journals that publish data arising from structural studies modulate their policies to take cognizance of new improved methodologies. The PDB and journals have evolved an accepted protocol to ensure the integrity of crystallographic results. As a result, the quality of available data and interpretations are becoming better over the years. However, there have been periodic efforts by some individuals who misuse validation mechanisms to selectively target published research through spurious challenges. These actions do more harm to the field of structural biology and runs counter to their claim to cleanse the system. The scientific systems in structural biology are robust and capable of self-correction and unwarranted vigilantism is counterproductive.

biophysics↗

bioRxiv2017

Harnessing optogenetics to probe sub-cellular mechanics

The mechanics of the actin cytoskeleton regulates cell morphogenesis during essential physiological processes. While cellular contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is not well understood. Here we combine optogenetic control of RhoA in space and time, live cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation in adherent fibroblasts. Local activation of RhoA not only stimulates local recruitment of actin and myosin, but also leads to increased traction forces outside the activation region that rapidly propagate across the cell via stress fibers and drive actin flow towards the region of heightened RhoA. Surprisingly, the flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fiber mechanics, as stress fibers are fluid-like without flow reversal in its absence. These experimental data are used to constrain a physical model, which demonstrates that stress fibers are elastic-like, even at time scales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulation of morphogenic events.

biophysics↗

bioRxiv2017

Dynactin has two antagonistic regulatory domains and exerts opposing effects on dynein motility

Dynactin is a dynein-regulating protein that increases the processivity of dynein movement on microtubules. Recent studies have shown that a tripartite complex of dynein-dynactin-Bicaudal D2 is essential for highly processive movement. To elucidate the regulation of dynein motility by dynactin, we focused on two isoforms (A and B) of dynactin 1 (DCTN1), the largest subunit of dynactin that contains both microtubule- and dynein-binding domains. The only difference between the primary structures of the two isoforms is that DCTN1B lacks the K-rich domain, a cluster of basic residues. We measured dynein motility by single molecule observation of recombinant dynein and dynactin. Whereas the tripartite complex containing DCTN1A exhibited highly processive movement, the complex containing DCTN1B dissociated from microtubules with no apparent processive movement. This inhibitory effect of DCTN1B was caused by reductions of the microtubule-binding affinities of both dynein and dynactin, which is attributed to the coiled-coil 1 domain of DCTN1. In DCTN1A, the K-rich domain antagonized these inhibitory effects. Therefore, dynactin has two antagonistic domains and promotes or suppresses dynein motility to accomplish correct localization and functions of dynein within a cell.

biophysics↗

bioRxiv2017

An allosteric theory of transcription factor induction

Allosteric regulation is found across all domains of life, yet we still lack simple, predictive theories that directly link the experimentally tunable parameters of a system to its input-output response. To that end, we present a general theory of allosteric transcriptional regulation using the Monod-Wyman-Changeux model. We rigorously test this model using the ubiquitous simple repression motif in bacteria by first predicting the behavior of strains that span a large range of repressor copy numbers and DNA binding strengths and then constructing and measuring their response. Our model not only accurately captures the induction profiles of these strains but also enables us to derive analytic expressions for key properties such as the dynamic range and [EC50]. Finally, we derive an expression for the free energy of allosteric repressors which enables us to collapse our experimental data onto a single master curve that captures the diverse phenomenology of the induction profiles.

biophysics↗