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bioRxiv2016

Histone code and higher-order chromatin folding: A hypothesis

Histone modifications alone or in combination are thought to modulate chromatin structure and function; a concept termed histone code. By combining evidence from several studies, we investigated if the histone code can play a role in higher-order folding of chromatin. Firstly using genomic data, we analyzed associations between histone modifications at the nucleosome level. We could dissect the composition of individual nucleosomes into five predicted clusters of histone modifications. Secondly, by assembling the raw reads of histone modifications at various length scales, we noticed that the histone mark relationships that exist at nucleosome level tend to be maintained at the higher orders of chromatin folding. Recently, a high-resolution imaging study showed that histone marks belonging to three of the five predicted clusters show structurally distinct and anti-correlated chromatin domains at the level of chromosomes. This made us think that the histone code can have a significant impact in the overall compaction of DNA: at the level of nucleosomes, at the level of genes, and finally at the level of chromosomes. As a result, in this article, we put forward a theory where the histone code drives not only the functionality but also the higher-order folding and compaction of chromatin.

biophysics↗

bioRxiv2016

Flowtrace: simple visualization of coherent structures in biological fluid flows

1We present a simple, intuitive algorithm for visualizing time-varying flow fields that can reveal complex flow structures with minimal user intervention. We apply this technique to a variety of biological systems, including the swimming currents of invertebrates and the collective motion of swarms of insects. We compare our results to more experimentally-diffcult and mathematically-sophisticated techniques for identifying patterns in fluid flows, and suggest that our tool represents an essential \"middle ground\" allowing experimentalists to easily determine whether a system exhibits interesting flow patterns and coherent structures without the need to resort to more intensive techniques. In addition to being informative, the visualizations generated by our tool are often striking and elegant, illustrating coherent structures directly from videos without the need for computational overlays. Our tool is available as fully-documented open-source code available for MATLAB, Python, or ImageJ at www.flowtrace.org.

biophysics↗

bioRxiv2016

Bridging microscopic and macroscopic mechanisms of p53-MDM2 binding using molecular simulations and kinetic network models

Under normal cellular conditions, the tumor suppressor protein p53 is kept at a low levels in part due to ubiquitination by MDM2, a process initiated by binding of MDM2 to the intrinsically disordered transactivation domain (TAD) of p53. Although many experimental and simulation studies suggest that disordered domains such as p53 TAD bind their targets nonspecifically before folding to a tightly-associated conformation, the molecular details are unclear. Toward a detailed prediction of binding mechanism, pathways and rates, we have performed large-scale unbiased all-atom simulations of p53-MDM2 binding. Markov State Models (MSMs) constructed from the trajectory data predict p53 TAD peptide binding pathways and on-rates in good agreement with experiment. The MSM reveals that two key bound intermediates, each with a non-native arrangement of hydrophobic residues in the MDM2 binding cleft, control the overall on-rate. Using microscopic rate information from the MSM, we parameterize a simple four-state kinetic model to (1) determine that induced-fit pathways dominate the binding flux over a large range of concentrations, and (2) predict how modulation of residual p53 helicity affects binding, in good agreement with experiment. These results suggest new ways in which microscopic models of bound-state ensembles can be used to understand biological function on a macroscopic scale.\n\nAUTHOR SUMMARYMany cell signaling pathways involve protein-protein interactions in which an intrinsically disordered peptide folds upon binding its target. Determining the molecular mechanisms that control these binding rates is important for understanding how such systems are regulated. In this paper, we show how extensive all-atom simulations combined with kinetic network models provide a detailed mechanistic understanding of how tumor suppressor protein p53 binds to MDM2, an important target of new cancer therapeutics. A simple four-state model parameterized from the simulations shows a binding-then-folding mechanism, and recapitulates experiments in which residual helicity boosts binding. This work goes beyond previous simulations of small-molecule binding, to achieve pathways and binding rates for a large peptide, in good agreement with experiment.

biophysics↗

bioRxiv2016

A Critical-like Collective State Leads to Long-range Cell Communication in Dictyostelium discoideum Aggregation

The transition from single-cell to multicellular behavior is important in early development but rarely studied. The starvation-induced aggregation of the social amoeba Dictyostelium discoideum into a multicellular slug is known to result from single-cell chemotaxis towards emitted pulses of cyclic adenosine monophosphate (cAMP). However, how exactly do transient short-range chemical gradients lead to coherent collective movement at a macroscopic scale? Here, we developed a multiscale model verified by quantitative microscopy to describe wide-ranging behaviors from chemotaxis and excitability of individual cells to aggregation of thousands of cells. To better understand the mechanism of long-range cell-cell communication and hence aggregation, we analyzed cell-cell correlations, showing evidence of self-organization at the onset of aggregation (as opposed to following a leader cell). Surprisingly, cell collectives, despite their finite size, show features of criticality known from phase transitions in physical systems. By comparing wild-type and mutant cells with impaired aggregation, we found the longest cellcell communication distance in wild-type cells, suggesting that criticality provides an adaptive advantage and optimally sized aggregates for the dispersal of spores.\n\nAuthor SummaryCells are often coupled to each other in cell collectives, such as aggregates during early development, tissues in the developed organism, and tumors in disease. How do cells communicate over macroscopic distances much larger than the typical cell-cell distance to decide how they should behave? Here, we developed a multiscale model of social amoeba, spanning behavior from individuals to thousands of cells. We show that local cell-cell coupling via secreted chemicals may be tuned to a critical value, resulting in emergent long-range communication and heightened sensitivity. Hence, these aggregates are remarkably similar to bacterial biofilms and neuronal networks, all communicating in a pulse-like fashion. Similar organizing principles may also aid our understanding of the remarkable robustness in cancer development.

biophysics↗

bioRxiv2016

Computing structure-based lipid accessibility of membrane proteins with mp_lipid_acc in RosettaMP

BackgroundMembrane proteins are vastly underrepresented in structural databases, which has led to a lack of computational tools and the corresponding inappropriate use of tools designed for soluble proteins. For membrane proteins, lipid accessibility is an essential property. Even though programs are available for sequence-based prediction of lipid accessibility and structure-based identification of solvent-accessible surface area, the latter does not distinguish between water accessible and lipid accessible residues in membrane proteins.\n\nResultsHere we present mp_lipid_acc, the first method to identify lipid accessible residues from the protein structure, implemented in the RosettaMP framework and available as a webserver. Our method uses protein structures transformed in membrane coordinates, for instance from PDBTM or OPM databases, and a defined membrane thickness to classify lipid accessibility of residues. mp_lipid_acc is applicable to both -helical and {beta}-barrel membrane proteins of diverse architectures with or without water-filled pores and uses a concave hull algorithm for classification. We further provide a manually curated benchmark dataset, on which our method achieves prediction accuracies of 90%.\n\nConclusionWe present a novel tool to classify lipid accessibility from the protein structure, which is applicable to proteins of diverse architectures and achieves prediction accuracies of 90% on a manually curated database. mp_lipid_acc is part of the Rosetta software suite, available at www.rosettacommons.org. The webserver is available at http://rosie.graylab.jhu.edu/mp_lipid_acc/submit and the benchmark dataset is available at http://tinyurl.com/mp-lipid-acc-dataset.\n\nSupplementary informationSupplementary information is available at BMC Bioinformatics.

biophysics↗

bioRxiv2016

Balance of microtubule stiffness and cortical tension determines the size of blood cells with marginal band across species

The fast blood stream of animals is associated with large shear stresses. Consequently, blood cells have evolved a special morphology and a specific internal architecture allowing them to maintain their integrity over several weeks. For instance, non-mammalian red blood cells, mammalian erythroblasts and platelets have a peripheral ring of microtubules, called the marginal band, that flattens the overall cell morphology by pushing on the cell cortex. In this article, we model how the shape of these cells stems from the balance between marginal band elasticity and cortical tension. We predict that the diameter of the cell scales with the total microtubule polymer, and verify the predicted law across a wide range of species. Our analysis also shows that the combination of the marginal band rigidity and cortical tension increases the ability of the cell to withstand forces without deformation. Finally, we model the marginal band coiling that occurs during the disc-to-sphere transition observed for instance at the onset of blood platelet activation. We show that when cortical tension increases faster than crosslinkers can unbind, the marginal band will coil, whereas if the tension increases slower, the marginal band may shorten as microtubules slide relative to each other.\n\nSignificance StatementMany blood cells have a discoidal shape, which is essential for them to function properly within the organism. For some cells, such as blood platelets, this shape is due to the interplay between the elasticity of the marginal band, which is a closed ring of stiff filaments called microtubules, and the tension of the cell cortex, a polymer scaffold associated with the plasma membrane. Dmitrieff et al. examined how cell size is determined by the mechanical balance between these two components. Remarkably, the theory is confirmed over nearly three orders of magnitudes, by data collected from 25 species. The theory also shows how the composite structure is adapted to resist transient mechanical challenges, as encountered in the blood stream.

biophysics↗

bioRxiv2016

Refractive index tomograms and dynamic membrane fluctuations of red blood cells from patients with diabetes mellitus

In this paper we present the optical characterisations of diabetic red blood cells (RBCs) in a non-invasive manner employing three-dimensional (3-D) quantitative phase imaging. By measuring 3-D refractive index tomograms and 2-D time-series phase images, the morphological (volume, surface area and sphericity), biochemical (haemoglobin concentration and content) and mechanical (membrane fluctuation) parameters were quantitatively retrieved at the individual cell level. With simultaneous measurements of individual cell properties, systematic correlative analyses on retrieved RBC parameters were also performed. Our measurements show that diabetic patients had RBCs of reduced cell sphericity and elevated intracellular haemoglobin concentration and content compared to healthy (non-diabetic) subjects. Furthermore, membrane deformability of diabetic RBCs is significantly lower than that of healthy, non-diabetic RBCs. Interestingly, non-diabetic RBCs exhibit strong correlations between the elevated glycated haemoglobin in RBC cytoplasm and decreased cell deformability, whereas diabetic RBCs do not show correlations. Our observations strongly support the idea that slow and irreversible glycation of haemoglobin and membrane proteins of RBCs by hyperglycaemia significantly compromises RBC deformability in diabetic patients.

biophysics↗

bioRxiv2016

Cryo-EM structure of haemoglobin at 3.2 A determined with the Volta phase plate

With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way1. These cameras are superior to previous detectors in coping with the intrinsically low contrast of radiation-sensitive organic materials embedded in amorphous ice, and so they have enabled the structure determination of several macromolecular assemblies to atomic or near-atomic resolution. According to one theoretical estimation, a few thousand images should suffice for calculating the structure of proteins as small as 17 kDa at 3 [A] resolution2. In practice, however, we are still far away from this theoretical ideal. Thus far, protein complexes that have been successfully reconstructed to high-resolution by single particle analysis (SPA) have molecular weights of ~100 kDa or larger3. Here, we report the use of Volta phase plate in determining the structure of human haemoglobin (64 kDa) at 3.2 [A]. Our results demonstrate that this method can be applied to complexes that are significantly smaller than those previously studied by conventional defocus-based approaches. Cryo-EM is now close to becoming a fast and cost-effective alternative to crystallography for high-resolution protein structure determination.

biophysics↗

bioRxiv2016

Slow and steady wins the race: encounters of myosin-5 and myosin-6 on shared actin filaments

In the cellular environment multiple myosins use the same filamentous actin (F-actin) tracks, yet little is known about how this track sharing is achieved and maintained. To assess the influence that different myosin classes have on each other, we developed an assay that combines two dynamic elements: elongating actin filaments with identified barbed and pointed ends, and myosins moving along these filaments. We studied two different myosins, myosin-5 and myosin-6. These myosins have distinct functions in the cell and are known to travel in opposite directions along actin filaments. Myosin-5 walks towards the barbed end of F-actin and generally into dynamically rearranging actin at the cell periphery. Myosin-6 is a pointed-end directed myosin that generally walks towards the cell center. We successfully reconstituted simultaneous bidirectional motility of myosin-5 and myosin-6 on single polymerizing filaments of actin. We report and provide statistical analysis of encounters between myosin-5 and myosin-6 walking along the single filaments. When myosin-5 and myosin-6 collide, myosin-5 detaches more frequently than myosin-6. The experimental observations are consistent with a stochastic stepping model based upon known myosin kinetics, which suggests that faster motors are more likely to detach.

biophysics↗

bioRxiv2016

In vivo imaging of coral tissue and skeleton with optical coherence tomography

Optical coherence tomography (OCT) is a non-invasive three-dimensional imaging technique with micrometer resolution allowing microstructural characterization of tissues in vivo and in real time. We present the first application of OCT for in vivo imaging of tissue and skeleton structure of intact living corals spanning a variety of morphologies and tissue thickness. OCT visualized different coral tissue layers (e.g. endoderm vs ectoderm), special structures such as mesenterial filaments and skeletal cavities, as well as mucus release from living corals. We also developed a new approach for non-invasive imaging and quantification of chromatophores containing green fluorescent protein (GFP)-like host pigment granules in coral tissue. The chromatophore system is hyper-reflective and can thus be imaged with good optical contrast in OCT, enabling quantification of chromatophore size, distribution and abundance. Because of its rapid imaging speed, OCT can also be used to quantify coral tissue movement showing that maximal linear contraction velocity was ~120 m per second upon high light stimulation. Based on OCT imaging of tissue expansion and contraction, we made first estimates of dynamic changes in the coral tissue surface area, which varied by a factor of 2 between the contracted and expanded state of the coral Pocillopora damicornis. We conclude that OCT is an excellent novel tool for in vivo tomographic imaging of corals that can reveal tissue and skeleton organization as well as quantify dynamic changes in tissue structure and coral surface area non-invasively and at high spatio-temporal resolution.

biophysics↗

bioRxiv2016

How fast can fast-folding proteins autonomously fold in silico?

In reported microcanonical molecular dynamics simulations, fast-folding proteins CLN025 and Trp-cage autonomously folded to experimentally determined native conformations. However, the folding times of these proteins derived from the simulations were more than 4-10 times longer than their experimental values. This article reports autonomous folding of CLN025 and Trp-cage in isobaric-isothermal molecular dynamics simulations with agreements within factors of 0.69-1.75 between simulated and experimental folding times at different temperatures. These results show that CLN025 and Trp-cage can now autonomously fold in silico as fast as in experiments, and suggest that the accuracy of folding simulations for fast-folding proteins begins to overlap with the accuracy of folding experiments. This opens new prospects of developing computer algorithms that can predict both ensembles of conformations and their interconversion rates for a protein from its sequence for artificial intelligence on how and when a protein acts as a receiver, switch, and relay to facilitate various subcellular-to-tissue communications. Then the genetic information that encodes proteins can be better read in the context of intricate biological functions.

biophysics↗

bioRxiv2016

Raoult’s law revisited: accurately predicting equilibrium relative humidity points for humidity control experiments

The humidity surrounding a sample is an important variable in scientific experiments. Biological samples in particular require not just a humid atmosphere but often a relative humidity (RH) that is in equilibrium with a stabilizing solution required to maintain the sample in the same state during measurements. The controlled dehydration of macromolecular crystals can lead to significant increases in crystal order, which often leads to higher diffraction quality. Devices that can accurately control the humidity surrounding crystals on a beamline have led to this technique being increasingly adopted, as experiments become easier and more reproducible. Matching the relative humidity to the mother liquor is the first step to allow the stable mounting of a crystal. In previous work, we measured the equilibrium relative humidity for a range of concentrations of the most commonly used precipitants and showed how this related to Raoults law for the equilibrium vapour pressure of water above a solution. However, a discrepancy between measured values and those predicted by theory could not be explained. Here, we have used a more precise humidity control device to determine equilibrium relative humidity points. The new results are in agreement with Raoults law. We also present a simple argument in statistical mechanics demonstrating that the saturated vapour pressure of a solvent is proportional to its mole fraction in an ideal solution: Raoults Law. The same argument can be extended to the case where solvent and solute molecules are of different size, as is the case with polymers. The results provide a framework for the correct maintenance of the RH surrounding samples.\n\nSynopsisThe equilibrium relative humidity values for a number of the most commonly used precipitants in biological macromolecule crystallisation have been measured using a new humidity control device. A simple argument in statistical mechanics demonstrates that the saturated vapour pressure of a solvent is proportional to its mole fraction in an ideal solution (Raoults Law). The same argument can be extended to the case where solvent and solute molecules are of different size.

biophysics↗

bioRxiv2016

Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots

The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. Cytoskeletal organization and dynamics are known to modulate cytoplasmic transport processes, but how local transport dynamics depends on the highly heterogeneous intracellular organization of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for transport dynamics in different sub-cellular environments. Rapid imaging of non-functionalized QDs revealed two populations with a 100-fold difference in diffusion constant. Depolymerization of actin increased the fast diffusing fraction, suggesting that slow QDs are trapped inside the actin network. When nanobody-functionalized QDs were targeted to different kinesin motor proteins and moved over microtubules, they did not experience strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 bound QDs displayed subtle directional fluctuations, because the specific subset of stable microtubules used by this motor underwent prominent undulations. Using actin-targeting agents we found that the actin network suppresses most microtubule shape remodeling, rather than promoting them. These results demonstrate how the spatial and structural heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics.

biophysics↗

bioRxiv2016

Charge influences substrate recognition and self-assembly of hydrophobic FG sequences

The nuclear pore complex controls the passage of molecules via hydrophobic phenylalanine-glycine (FG) domains on nucleoporins. Such FG-domains consist of repeating units of FxFG, FG, or GLFG sequences, which can be interspersed with highly charged amino acid sequences. Despite the high density of charge exhibited in certain FG-domains, if and how charge influences FG-domain self-assembly and selective binding of nuclear transport receptors is largely unexplored. Studying how individual charged amino acids contribute to nuclear pore selectivity is challenging with modern in vivo and in vitro techniques due to the complexity of nucleoporin sequences. Here, we present a rationally designed approach to deconstruct essential components of nucleoporins down to 14 amino acid sequences. With these nucleoporin-based peptides, we systematically dissect how charge type and placement of charge influences self-assembly and selective binding of FG-containing gels. Specifically, we find that charge type determines which hydrophobic substrates FG sequences recognize while spatial localization of charge tunes hydrophobic self-assembly and receptor selectivity of FG sequences.

biophysics↗

bioRxiv2016

Voltage-dependent K+ Channels Improve the Energy Efficiency of Signalling in Blowfly Photoreceptors

Voltage-dependent conductances in many spiking neurons are tuned to reduce action potential energy consumption, so improving the energy efficiency of spike coding. However, the contribution of voltage-dependent conductances to the energy efficiency of analogue coding, by graded potentials in dendrites and non-spiking neurons, remains unclear. We investigate the contribution of voltage-dependent conductances to the energy efficiency of analogue coding by modelling blowfly R1-6 photoreceptor membrane. Two voltage-dependent delayed rectifier K+ conductances (DRs) shape the membrane's voltage response and contribute to light adaptation. They make two types of energy saving. By reducing membrane resistance upon depolarisation they convert the cheap, low bandwidth membrane needed in dim light to the expensive high bandwidth membrane needed in bright light. This investment of energy in bandwidth according to functional requirements can halve daily energy consumption. Second, DRs produce negative feedback that reduces membrane impedance and increases bandwidth. This negative feedback allows an active membrane with DRs to consume at least 30% less energy than a passive membrane with the same capacitance and bandwidth. Voltage gated conductances in other non-spiking neurons, and in dendrites, might be organized to make similar savings.

biophysics↗

bioRxiv2016

Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality

Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5 [A] or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5 [A], while the resolution of the map reached 4.0 [A] for the same number of particles, when the estimated resolution of streptavidin crystal was 4 [A] or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6 [A], a marked improvement over the value of 3.9 [A] obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.

biophysics↗

bioRxiv2016

Revealing compartmentalised membrane diffusion in living cells with interferometric scattering microscopy

Single-particle tracking is a powerful tool for studying single molecule behaviour involving plasma membrane-associated events in cells. Here, we show that interferometric scattering microscopy (iSCAT) combined with gold nanoparticle labeling can be used to follow the motion of membrane proteins in the plasma membrane of live cultured mammalian cell lines and hippocampal neurons. The unique combination of microsecond temporal resolution and nanometer spatial precision reveals signatures of a compartmentalised plasma membrane in neurons.

biophysics↗

bioRxiv2016

OpenMM 7: Rapid Development of High Performance Algorithms for Molecular Dynamics

OpenMM is a molecular dynamics simulation toolkit with a unique focus on extensibility. It allows users to easily add new features, including forces with novel functional forms, new integration algorithms, and new simulation protocols. Those features automatically work on all supported hardware types (including both CPUs and GPUs) and perform well on all of them. In many cases they require minimal coding, just a mathematical description of the desired function. They also require no modification to OpenMM itself and can be distributed independently of OpenMM. This makes it an ideal tool for researchers developing new simulation methods, and also allows those new methods to be immediately available to the larger community.

biophysics↗