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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2016

Resolving Biology Beyond the Diffraction Limit with Single-Molecule Localization Microscopy

Optical imaging provides a window into the microscopic world, but the level of observable detail is ultimately limited by the wavelength of light being employed. By harnessing the physics of photoswitchable dyes and fluorescent proteins, single-molecule localization microscopy (SMLM) provides a window into the nano-world of biology. This mini-review article provides a short overview of SMLM and discusses some of its prospects for the future.

biophysics↗

bioRxiv2016

Quantifying the Heat Dissipation from Molecular Motor’s Transport Properties in Nonequilibrium Steady States

Theoretical analysis, which maps single molecule time trajectories of a molecular motor onto unicyclic Markov processes, allows us to evaluate the heat dissipated from the motor and to elucidate its dependence on the mean velocity and diffusivity. Unlike passive Brownian particles in equilibrium, the velocity and diffusion constant of molecular motors are closely inter-related to each other. In particular, our study makes it clear that the increase of diffusivity with the heat production is a natural outcome of active particles, which is reminiscent of the recent experimental premise that the diffusion of an exothermic enzyme is enhanced by the heat released from its own catalytic turnover. Compared with freely diffusing exothermic enzymes, kinesin-1 whose dynamics is confined on one-dimensional tracks is highly efficient in transforming conformational fluctuations into a locally directed motion, thus displaying a significantly higher enhancement in diffusivity with its turnover rate. Putting molecular motors and freely diffusing enzymes on an equal footing, our study offers thermodynamic basis to understand the heat enhanced self-diffusion of exothermic enzymes.

biophysics↗

bioRxiv2016

Active Vertex Model for Cell-Resolution Description of Epithelial Tissue Mechanics

We introduce an Active Vertex Model (AVM) for cell-resolution studies of the mechanics of confluent epithelial tissues consisting of tens of thousands of cells, with a level of detail inaccessible to similar methods. The AVM combines the Vertex Model for confluent epithelial tissues with active matter dynamics. This introduces a natural description of the cell motion and accounts for motion patterns observed on multiple scales. Furthermore, cell contacts are generated dynamically from positions of cell centres. This not only enables efficient numerical implementation, but provides a natural description of the T1 transition events responsible for local tissue rearrangements. The AVM also includes cell alignment, cell-specific mechanical properties, cell growth, division and apoptosis. In addition, the AVM introduces a flexible, dynamically changing boundary of the epithelial sheet allowing for studies of phenomena such as the fingering instability or wound healing. We illustrate these capabilities with a number of case studies.

biophysics↗

bioRxiv2016

New Insights into the Dynamics of Swarming Bacteria: A Theoretical Study

In the present work we simulate the basic two-dimensional dynamics of swarming E. coli bacteria on the surface of a moderately soft agar plate. Individual bacteria are modelled by self-propelled ridged bodies (agents), which interact with each other only through inelastic collision and with the highly viscous environment through damping forces. The motion of single agents is modelled closely corresponding to the behaviour of swimming bacteria. The dynamics of the model were adjusted to reproduce the experimental measurements of swimming E. coli K-12. Accordingly, simulations with loosely packed agents ({rho}{approx}0) show typical run-and-tumble statistics. In contrast, simulations with densely packed agents ({rho}{approx}0.3-0.7) are dominated by interactions (collisions) between agents which lead to the emergence of swarming behaviour. In addition, we model the motion of single agents on the base of modified run-and-tumble dynamics, where the bacteria do not turn actively during the tumble. We show that simulations with densely packed modified agents lead as well the emergence of swarming behaviour, if rotational diffusion is considered.

biophysics↗

bioRxiv2016

Single-enzyme approach predicts natural emergence of inhibitor-activator duality

The classical theory of enzymatic inhibition aims to quantitatively describe the effect of certain molecules--called inhibitors--on the progression of enzymatic reactions, but growing signs indicate that it must be revised to keep pace with the single-molecule revolution that is sweeping through the sciences. Here, we take the single enzyme perspective and rebuild the theory of enzymatic inhibition from the bottom up. We find that accounting for multi-conformational enzyme structure and intrinsic randomness cannot undermine the validity of classical results in the case of competitive inhibition; but that it should strongly change our view on the uncompetitive and mixed modes of inhibition. There, stochastic fluctuations on the single-enzyme level could give rise to inhibitor-activator duality--a phenomenon in which, under some conditions, the introduction of a molecule whose binding shuts down enzymatic catalysis will counter intuitively work to facilitate product formation. We state--in terms of experimentally measurable quantities--a mathematical condition for the emergence of inhibitor-activator duality, and propose that it could explain why certain molecules that act as inhibitors when substrate concentrations are high elicit a non-monotonic dose response when substrate concentrations are low. The fundamental and practical implications of our findings are thoroughly discussed.

biophysics↗

bioRxiv2016

The Liquid Structure of Elastin

The protein elastin imparts extensibility, elastic recoil, and resilience to tissues including arterial walls, skin, lung alveoli, and the uterus. Elastin and elastin-like peptides are intrinsically disordered hydrophobic proteins that undergo liquid-liquid phase separation upon self-assembly. Despite extensive study for over eighty years, the structure of elastin remains controversial. We use molecular dynamics simulations on a massive scale to elucidate the structural ensemble of aggregated elastin-like peptides. Consistent with the entropic nature of elastic recoil, the aggregated state is stabilized both by the hydrophobic effect and by conformational entropy. The polypeptide backbone forms transient, sparse hydrogen-bonded turns and remains significantly hydrated even as self-assembly triples the extent of nonpolar side-chain contacts. The assembly approaches a maximally-disordered, melt-like state, which may be called the liquid state of proteins. These findings resolve long-standing controversies regarding elastin structure and function and afford insight of broad relevance to the phase separation of disordered proteins.

biophysics↗

bioRxiv2016

Cell Mechanosensing and Avoidance of Strain Gradients

Cyclically stretched cells are known to exhibit a strain-induced reorientation response. In this study, we comprehensively analyse this behaviour for human fibroblasts subjected to a highly non-uniform strain field within a polymethylsiloxane microdevice. We demonstrate a strong correlation between the strain amplitude and the degree of cell alignment perpendicular to the principal strain direction (stretching avoidance). Analogously, our results indicate that the strain gradient amplitude and direction also regulate this reorientation through a coordinated gradient avoidance response. We stipulate that strain gradients are thus biologically relevant mechanical cues sensed by cells. To gain insight into the underlying mechanosensing processes, we also studied focal adhesion reorganization and the effect of modulating myosin-II contractility. The extracted focal adhesion orientation distributions are similar to those obtained for the cell bodies, and their density is increased by the presence of stretching forces. Moreover, it was found that the myosin-II activity promoter calyculin-A has little effect on the cellular response, while the inhibitor blebbistatin suppresses cell and focal adhesion alignment and reduces focal adhesion density. These results confirm that similar internal structures involved in sensing and responding to strain direction and amplitude are also key players in strain gradient mechanosensing and avoidance.

biophysics↗

bioRxiv2016

Non-equilibrium chromosome looping via molecular slip-links

We propose a model for the formation of chromatin loops based on the diffusive sliding of a DNA-bound factor which can dimerise to form a molecular slip-link. Our slip-links mimic the behaviour of cohesin-like molecules, which, along with the CTCF protein, stabilize loops which organize the genome. By combining 3D Brownian dynamics simulations and 1D exactly solvable non-equilibrium models, we show that diffusive sliding is sufficient to account for the strong bias in favour of convergent CTCF-mediated chromosome loops observed experimentally. Importantly, our model does not require any underlying, and energetically costly, motor activity of cohesin. We also find that the diffusive motion of multiple slip-links along chromatin may be rectified by an intriguing ratchet effect that arises if slip-links bind to the chromatin at a preferred \"loading site\". This emergent collective behaviour is driven by a 1D osmotic pressure which is set up near the loading point, and favours the extrusion of loops which are much larger than the ones formed by single slip-links.

biophysics↗

bioRxiv2016

Assembly of photoactive Orange Carotenoid Protein from its domains unravels a carotenoid shuttle mechanism

The Orange Carotenoid Protein (OCP) is indispensable for cyanobacterial photoprotection by quenching phycobilisome fluorescence upon photoconversion from the orange OCPO to the red OCPR form. Cyanobacterial genomes frequently harbor, besides genes for Orange Carotenoid Proteins (OCPs), several genes encoding homologs of OCPs N- or C-terminal domains (NTD, CTD). Unlike the well-studied NTD homologs, called Red Carotenoid Proteins (RCPs), the role of CTD homologs remains elusive. We show how OCP can be reassembled from its functional domains. Expression of Synechocystis OCP-CTD in carotenoid-producing Escherichia coli yielded violet-colored proteins, which, upon mixing with the RCP-apoprotein, produced an orange-like photoswitchable form that further photoconverted into a species spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. The CTD itself is a novel, dimeric carotenoid-binding protein, which effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein, assigning physiological roles to CTD homologs and explaining the evolutionary process of OCP formation.\n\nOne Sentence SummaryThe C-domain of cyanobacterial OCP dimerizes, binds a carotenoid, and delivers it to the N-domain forming photoactive OCP.

biophysics↗

bioRxiv2016

Label-free high-resolution 3-D imaging of gold nanoparticles inside live cells using optical diffraction tomography

Delivery of gold nanoparticles (GNPs) into live cells has high potentials, ranging from molecular-specific imaging, photodiagnostics, to photothermal therapy. However, studying the long-term dynamics of cells with GNPs using conventional fluorescence techniques suffers from phototoxicity and photobleaching. Here, we present a method for 3-D imaging of GNPs inside live cells exploiting refractive index (RI) as imaging contrast. Employing optical diffraction tomography, 3-D RI tomograms of live cells with GNPs are precisely measured for an extended period with sub-micrometer resolution. The locations and contents of GNPs in live cells are precisely addressed and quantified due to their distinctly high RI values, which was validated by confocal fluorescence imaging of fluorescent dye conjugated GNPs. In addition, we perform quantitative imaging analysis including the segmentations of GNPs in the cytosol, the volume distributions of aggregated GNPs, and the temporal evolution of GNPs contents in HeLa and 4T1 cells.\n\nAbbreviations

biophysics↗

bioRxiv2017

Monitoring Microtubule Mechanical Vibrations via Optomechanical Coupling

The possible disruption of a microtubule during mitosis can control the duplication of a cancer cell. Cancer detection and treatment may be possible based on the detection and control of microtubule mechanical oscillations in cells throughexternal fields (e.g. electromagnetic or ultrasound). However, little is known about the dynamic (high-frequency) mechanical properties of microtubules. Here we propose to control the vibrations of a doubly clamped microtubule by tip electrodes and to detect its motion via the optomechanical coupling between the vibrational modes of the microtubule and an optical cavity. In the presence of a red-detuned strong pump laser, this coupling leads to optomechanical induced transparency of an optical probe field, which can be detected with state-of-the art technology. The center frequency and linewidth of the transparency peak give the resonance frequency and damping rate of the microtubule respectively, while the height of the peak reveals information about the microtubule-cavity field coupling. Our method should yield new knowledge about the physical properties of microtubules, which will enhance our capability to design physical cancer treatment protocols as alternatives to chemotherapeutic drugs.

biophysics↗

bioRxiv2017

Bacterial Conversations and Pattern Formation

It has long been recognized that certain bacterial groups exhibit cooperative behavioral patterns. Bacteria accomplish such communication via exchange of extracellular signaling molecules called pheromones(autoinducer or quorum sensing molecules). As the bacterial culture grows, signal molecules are released into extracellular milieu accumulate, changing water fluidity. Under such threshold conditions swimming bacterial suspensions impose a coordinated water movement on a length scale of the order 10 to 100 micrometers compared with a bacterial size of the order of 3 micrometers.Here, we investigate the non-local hydrodynamics of the quorum state and pattern formation using forced Burgers equation with Kwak transformation. Such approach resulted in the conversion of the Burgers equation paradigm into a reaction-diffusion system. The examination of the dynamics of the quorum sensing system, both analytically as well as numerically result in similar long-time dynamical behaviour.

biophysics↗

bioRxiv2017

How does Mg2+ modulate the RNA folding mechanism - a case study of G:C W:W Trans base pair.

Reverse Watson-Crick G:C base pairs (G:C W:W Trans) occur frequently in different functional RNAs. It is one of the few base pairs whose gas phase optimized isolated geometry is inconsistent with the corresponding experimental geometry. Several earlier studies indicate that accumulation of positive charge near N7 of guanine, through posttranscriptional modification, direct protonation or coordination with Mg2+, can stabilize the experimental geometry. Interestingly, recent studies reveal significant variation in the position of putatively bound Mg2+. This, in conjunction with recently raised doubts regarding some of the Mg2+ assignments near the imino nitrogen of guanine, is suggestive of the existence of multiple Mg2+ binding modes for this base pair. Our detailed investigation of Mg2+ bound G:C W:W Trans pairs, occurring in high resolution RNA crystal structures, show that they occur in 14 different contexts, 8 out of which display Mg2+ binding at the Hoogsteen edge of guanine. Further examination of occurrences in these 8 contexts led to the characterization of three different Mg2+ binding modes, (i) direct binding via N7 coordination, (ii) direct binding via O6 coordination and (iii) binding via hydrogen bonding interaction with the first shell water molecules. In the crystal structures, the latter two modes are associated with a buckled and propeller twisted geometry of the base pair. Interestingly, respective optimized geometries of these different Mg2+ binding modes (optimized at B3LYP) are consistent with their corresponding experimental geometries. Subsequent interaction energy calculations at MP2 level, and decomposition of its components, suggest that for G:C W:W Trans, Mg2+ binding can fine tune the base pair geometries without compromising with their stability. Our results, therefore, underline the importance of the mode of binding of Mg2+ ions in shaping RNA structure, folding and function.

biophysics↗

bioRxiv2017

Active diffusion and advection in the Drosophila ooplasm result from the interplay of the actin and microtubule cytoskeletons

Transport in cells occurs via a delicate interplay of passive and active processes, including diffusion, directed transport and advection. Despite progresses in super-resolution microscopy, discriminating and quantifying these processes is a challenge, requiring tracking of rapidly moving, sub-diffraction objects in a crowded, noisy environment. Here we use Differential Dynamic Microscopy with different contrast mechanisms to provide a thorough characterization of the dynamics in the Drosophila oocyte. We study the movement of vesicles and the elusive motion of a cytoplasmic F-actin mesh, a known regulator of cytoplasmic flows. We find that cytoplasmic motility constitutes a combination of directed motion and random diffusion. While advection is mainly attributed to microtubules, we find that active diffusion is driven by the actin cytoskeleton, although it is also enhanced by the flow. We also find that an important dynamic link exists between vesicles and cytoplasmic F-actin motion, as recently suggested in mouse oocytes.

biophysics↗

bioRxiv2017

Nucleotide-dependent stiffness suggests role of interprotofilament bonds in microtubule assembly

Many eukaryotic cell functions depend on dynamic instability, meaning the nucleotide-driven assembly and disassembly of microtubules. Assembly requires the constituent tubulin dimers to bind the nucleotide GTP, and its subsequent hydrolysis to GDP induces disassembly. The underlying structural mechanisms, however, are not well understood. Here, we determine the strength of contacts in the microtubule lattice by combining high precision measurements of the bending stiffness of analogues of GTP and GDP microtubules with a recent theoretical model. While previous structural studies have focussed on how the curvature of the tubulin dimer is affected by nucleotide binding, we present evidence of a dramatic regulation of the lateral interactions between the parallel protofilaments that dimers form in the microtubule. We conclude that the shear coupling between neighboring protofilaments is at least two orders of magnitude stronger in the GTP state than in the GDP state, and discuss the implications for the microtubule assembly.

biophysics↗

bioRxiv2017

Reverse Watson-Crick purine-purine base pairs : the Sharp-turn motif and other structural consequences in functional RNAs

Identification of static and/or dynamic roles of different noncanonical base pairs is essential for a comprehensive understanding of the sequence-structure-function space of RNA. In this context, reverse Watson-Crick purine-purine base pairs (A:A, G:G&A:GW:W Trans) constitute an interesting class of noncanonical base pairs in RNA due to their characteristic C1'-C1' distance (highest among all base pairing geometries) and parallel local strand orientation. Structural alignment of the RNA stretches containing these W:W Trans base pairs with their corresponding homologous sites in a non-redundant set of RNA crystal structures show that, as expected, these base pairs are associated with specific structural folds or functional roles. Detailed analysis of these contexts further revealed a bimodal distribution in the local backbone geometry parameters associated with these base pairs. One mode, populated by both A:A and G:G W:W Trans pairs, manifests itself as a characteristic backbone fold. We call this fold a Sharp-turn motif. The other mode is exclusively associated with A:A W:W Trans pairs involved in mediating higher order interactions. The same trend is also observed in available solution NMR structures. We have also characterized the importance of recurrent hydrogen bonding interactions between adenine and guanine in W:W Trans geometry. Quantum chemical calculations performed at M05-2X/6-31++(2d,2p) level explain how the characteristic electronic properties of these W:W Trans base pairs facilitate their occurrence in such exclusive structural folds that are important for RNA functionalities.

biophysics↗

bioRxiv2017

Structural landscape of base pairs containing post-transcriptional modifications in RNA

Base pairs involving post-transcriptionally modified nucleobases are believed to play important roles in a wide variety of functional RNAs. Here we present our attempts towards understanding the structural and functional role of naturally occurring modified base pairs by analyzing their distribution in different RNA classes, with the help of crystal structure and sequence database analyses. In addition, we quantify the variation in geometrical features of modified base pairs within RNA structures, and characterize their optimum geometries and binding energies using advanced quantum chemical methods. Further comparison of modified base pairs with their unmodified counterparts illustrates the effect of steric and electronic structure alterations due to base modifications. Analysis of specific structural contexts of modified base pairs in RNA crystal structures revealed several interesting scenarios, including those at the tRNA:rRNA interface, antibiotic-binding site and the three-way junctions within tRNA, which when analyzed in context of available experimental data, allowed us to correlate the occurrence and strength of modified base pairs with the specific functional roles they play in context of RNA macromolecules.

biophysics↗

bioRxiv2017

Accurate alignment between correlated light, cryo-light, and electron cryo-microscopy data using sample support features

Combining fluorescence microscopy with electron cryo-tomography allows, in principle, spatial localization of tagged macromolecular assemblies and structural features within the cellular environment. To allow precise localization and scale integration between the two disparate imaging modalities, accurate alignment procedures are needed. Here, we describe a marker-free method for aligning images from light or cryo-light fluorescence microscopy and from electron cryo-microscopy that takes advantage of sample support features, namely the holes in the carbon film. We find that the accuracy of this method, as judged by prediction errors of the hole center coordinates, is better than 100 nm.

biophysics↗