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bioRxiv2017

Spot-On: robust model-based analysis of single-particle tracking experiments

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce \"Spot-On\", an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.\n\nIMPACT STATEMENTSpot-On is an easy-to-use website that makes a rigorous and bias-corrected modeling framework for analysis of single-molecule tracking experiments available to all.

biophysics↗

bioRxiv2017

Symmetric, explicit numerical integrator for molecular dynamics equations of motion with a generalized friction

A general mathematical scheme to construct symmetric, explicit numerical integrators of Newtonian equations of motion endowed with a generalized friction is provided for molecular dynamics (MD) study. The exact integrations are done for all the decomposed vector fields, including the one that contains the friction term. On the basis of the symmetric composition scheme with the adjoint for the resulting maps, integrators with any local order of accuracy can be systematically constructed. Among them, the second order P2S1 integrator gives the least evaluation of atomic force and potential, which are most time consuming in MD simulations. As examples of the friction function, three functional types are considered: constant, Laurent polynomial, and exponential with respect to the kinetic energy. Several MD equations of motion fall into these categories, and the numerical examinations of their integrators using a basic model system give positive results on the accuracy and efficiency. The extended phase-space scheme also presents an invariant function, which allows to easily detect numerical errors in the integration process by monitoring the function value.

biophysics↗

bioRxiv2017

Liver physiological T1rho dynamics associated with age and gender

PurposeUsing a single breathhold black blood sequence, the current study aims to understand the physiological ranges of liver T1rho relaxation for women and men.\n\nMaterials and MethodsThis volunteer study was conducted with institutional ethics committee approval, and included 62 females (age mean: 38.9 years; range: 18-75 years) and 34 males (age mean: 44.7 years, range: 24-80 years). MRI was conducted with a 3.0 T scanner, with six spin-lock times of 0, 10, 20, 25, 35, 50msec and a single breathhold of 12 seconds. Six slices were acquired for each examination.\n\nResultsFemale liver T1rho value ranged between 35.07 to 51.97ms, showed an age-dependent decrease with younger women had a higher measurement. Male Liver T1rho values ranged between 34.94 to 43.39 ms, and there was no evidential age-dependence. For females, there was a trend that liver T1rho value could be 4%-5% lower during menstrual phase than nonmenstrual phase. For both females and males, no evidential association was seen between body mass index and liver T1rho.\n\nConclusionLiver T1rho physiological value for males have relatively narrow distribution, while physiological value for females have wider distribution, and decreases with age.\n\nKey points1. Liver T1rho shows an age-dependency in women, with young women showing higher measurement. This age-dependency of liver T1rho measurement is not evidential in men. Post-menopausal women have similar liver T1rho value as men.\n\n2. Women at menstrual phase may have slight lower liver T1rho measurement.\n\n3. No association was noted between body mass index and liver T1rho\n\n4. When blood signal suppression sequence is used, in a population of 62 healthy women and 34 healthy men, the highest measured liver T1rho was 52 msec for young women, 44.7 msec for post-menopausal women, and 43.4 msec for men.

biophysics↗

bioRxiv2017

Fast, robust and precise 3D localization for arbitrary point spread functions

We present a fitter for 3D single-molecule localization of arbitrary, experimental point spread functions (PSFs) that reaches minimum uncertainty for EMCCD and sCMOS cameras, and achieves more than 105 fits/s. We provide tools to robustly model experimental PSFs and correct for depth induced aberrations, which allowed us to achieve an unprecedented 3D resolution with engineered astigmatic PSFs, and acquire high quality 3D superresolution images even on standard microscopes without 3D optics.

biophysics↗

bioRxiv2017

Structural Basis of Mitochondrial Receptor Binding and GTP Driven Conformational Constriction by Dynamin-Related Protein 1

Mitochondrial inheritance, genome maintenance, and metabolic adaptation all depend on organelle fission by Dynamin-Related Protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogs Mitochondrial Dynamics 49 and 51 (MID49/MID51) and Mitochondrial Fission Factor (MFF), but the mechanisms by which these proteins recruit DRP1 and regulate its activities are unknown. Here we present a cryoEM structure of human, full-length DRP1 bound to MID49 and an analysis of structure- and disease-based mutations. We report that GTP binding allosterically induces a remarkable elongation and rotation of the G-domain, Bundle-Signaling Element (BSE) and connecting hinge loops of DRP1. In this nucleotide-bound conformation, a distributed network of multivalent interactions promotes DRP1 copolymerization into a linear filament with MID49, MID51 or both. Subsequent GTP hydrolysis and exchange within the filament leads to receptor dissociation, shortening through disassembly, and concomitant curling of DRP1 oligomers into closed rings. The dimensions of the closed DRP1 rings are consistent with DRP1-constricted mitochondrial tubules observed in human cells. These structures are the first views of full-length, receptor- and nucleotide-bound dynamin-family GTPases and--in comparison with nucleotide-free crystal structures--teach us how these molecular machines perform mechanical work through nucleotide-driven allostery.

biophysics↗

bioRxiv2017

Microbial mutualism at a distance

The exchange of diffusive metabolites is known to control the spatial patterns formed by microbial populations, as revealed by recent studies in the laboratory. However, the matrices used, such as agarose pads, lack the structured geometry of many natural microbial habitats, including in the soil or on the surfaces of plants or animals. Here we address the important question of how such geometry may control diffusive exchanges and microbial interaction. We model mathematically mutualistic interactions within a minimal unit of structure: two growing reservoirs linked by a diffusive channel through which metabolites are exchanged. The model is applied to study a synthetic mutualism, experimentally parameterised on a model algal-bacterial co-culture. Analytical and numerical solutions of the model predict conditions for the successful establishment of remote mutualisms, and how this depends, often counterintutively, on diffusion geometry. We connect our findings to understanding complex behaviour in synthetic and naturally occurring microbial communities.

biophysics↗

bioRxiv2017

De Novo Prediction of Human Chromosome Structures: Epigenetic Marking Patterns Encode Genome Architecture

Inside the cell nucleus, genomes fold into organized structures that are characteristic of cell type. Here, we show that this chromatin architecture can be predicted de novo using epigenetic data derived from ChIP-Seq. We exploit the idea that chromosomes encode a one-dimensional sequence of chromatin structural types. Interactions between these chromatin types determine the three-dimensional (3D) structural ensemble of chromosomes through a process similar to phase separation. First, a recurrent neural network is used to infer the relation between the epigenetic marks present at a locus, as assayed by ChIP-Seq, and the genomic compartment in which those loci reside, as measured by DNA-DNA proximity ligation (Hi-C). Next, types inferred from this neural network are used as an input to an energy landscape model for chromatin organization (MiChroM) in order to generate an ensemble of 3D chromosome conformations. After training the model, dubbed MEGABASE (Maximum Entropy Genomic Annotation from Biomarkers Associated to Structural Ensembles), on odd numbered chromosomes, we predict the chromatin type sequences and the subsequent 3D conformational ensembles for the even chromosomes. We validate these structural ensembles by using ChIP-Seq tracks alone to predict Hi-C maps as well as distances measured using 3D FISH experiments. Both sets of experiments support the hypothesis of phase separation being the driving process behind compartmentalization. These findings strongly suggest that epigenetic marking patterns encode sufficient information to determine the global architecture of chromosomes and that de novo structure prediction for whole genomes may be increasingly possible.

biophysics↗

bioRxiv2017

Energetics and Conformational Pathways of Functional Rotation in the Multidrug Transporter AcrB

The multidrug transporter AcrB transports a broad range of drugs out of the cell by means of the proton-motive force. The asymmetric crystal structure of trimeric AcrB suggests a functionally rotating mechanism for drug transport. Despite various supportive evidences from biochemical and simulation studies for this mechanism, the link between the functional rotation and proton translocation across the membrane remains elusive. Here, calculating the minimum free energy pathway of the functional rotation for the complete AcrB trimer, we describe the structural and energetic basis behind the coupling between the functional rotation and the proton translocation at atomic-level. Free energy calculations show that protonation of Asp408 in the transmembrane portion of the drug-bound protomer drives the functional rotation. The conformational pathway identifies vertical shear motions among several transmembrane helices, which regulates alternate access of water in the transmembrane as well as peristaltic motions pumping drugs in the periplasm.

biophysics↗

bioRxiv2017

Tau directs intracellular trafficking by regulating the forces exerted by kinesin and dynein teams

Organelles, proteins, and mRNA are transported bidirectionally along microtubules by plus-end directed kinesin and minus-end directed dynein motors. Microtubules are decorated by microtubule-associated proteins (MAPs) that organize the cytoskeleton, regulate microtubule dynamics and modulate the interaction between motor proteins and microtubules to direct intracellular transport. Tau is a neuronal MAP that stabilizes axonal microtubules and crosslinks them into bundles. Dysregulation of tau leads to a range of neurodegenerative diseases known as tauopathies including Alzheimers disease (AD). Tau reduces the processivity of kinesin and dynein by acting as an obstacle on the microtubule. Single-molecule assays indicate that kinesin-1 is more strongly inhibited than kinesin-2 or dynein, suggesting tau might act to spatially modulate the activity of specific motors. To investigate the role of tau in regulating bidirectional transport, we isolated phagosomes driven by kinesin-1, kinesin-2, and dynein and reconstituted their motility along microtubules. We find that tau biases bidirectional motility towards the microtubule minus-end in a dose-dependent manner. Optical trapping measurements show that tau increases the magnitude and frequency of forces exerted by dynein through inhibiting opposing kinesin motors. Mathematical modeling indicates that tau controls the directional bias of intracellular cargoes through differentially tuning the processivity of kinesin-1, kinesin-2, and dynein. Taken together, these results demonstrate that tau modulates motility in a motor-specific manner to direct intracellular transport, and suggests that dysregulation of tau might contribute to neurodegeneration by disrupting the balance of plus- and minus-end directed transport.\n\nSynopsis and Graphical Table of ContentsWe isolated endogenous cargoes, along with a complement of kinesin-1, kinesin-2, and dynein motors, and reconstituted their bidirectional motility in vitro. We find that tau, a microtubule-associated protein that stabilizes microtubules in neuronal axons, directs bidirectional cargoes towards the microtubule minus end by tuning the balance of forces exerted by kinesin and dynein teams. These results suggest a general mechanism for regulating the transport of intracellular cargoes through modulating the relative activity of opposing motor teams.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC=\"FIGDIR/small/173609_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (31K):\norg.highwire.dtl.DTLVardef@8ba4e0org.highwire.dtl.DTLVardef@1f5945corg.highwire.dtl.DTLVardef@1df2f47org.highwire.dtl.DTLVardef@198f5d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2017

Autocatalytic microtubule nucleation determines the size and mass of spindles

Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation (1, 2). Previous evidence suggests that microtubules nucleate throughout the spindle structure (3-5). However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. Thus, the size of spindles is determined by the distance where one microtubule nucleates on average less than one new microtubule. This mechanism provides an upper limit to spindle size even when resources are not limiting and may have implications for spindle scaling during development (6, 7).

biophysics↗

bioRxiv2017

Gpufit: An open-source toolkit for GPU-accelerated curve fitting

We present a general purpose, open-source software library for estimation of nonlinear parameters by the Levenberg-Marquardt algorithm. The software, Gpufit, runs on a Graphics Processing Unit (GPU) and executes computations in parallel, resulting in a significant gain in performance. We measured a speed increase of up to 42 times when comparing Gpufit with an identical CPU-based algorithm, with no loss of precision or accuracy. Gpufit is designed such that it is easily incorporated into existing applications or adapted for new ones. Multiple software interfaces, including to C, Python, and Matlab, ensure that Gpufit is accessible from most programming environments. The full source code is published as an open source software repository, making its function transparent to the user and facilitating future improvements and extensions. As a demonstration, we used Gpufit to accelerate an existing scientific image analysis package, yielding significantly improved processing times for super-resolution fluorescence microscopy datasets.

biophysics↗

bioRxiv2017

A catch-bond drives stator mechanosensitivity in the Bacterial Flagellar Motor

The bacterial flagellar motor (BFM) is the rotary motor which powers the swimming and swarming of many motile bacteria. The torque is provided by stator units, ion motive force powered ion channels known to assemble and disassemble dynamically in the BFM. This turnover is mechano-sensitive, with the number of engaged units dependent upon the viscous load experienced by the motor through the flagellum. However, the molecular mechanism driving BFM mechano-sensitivity is unknown. Here we directly measure the kinetics of arrival and departure of the stator units in individual wild-type motors via analysis of high-resolution recordings of motor speed, while dynamically varying the load on the motor via external magnetic torque. Obtaining the real-time stator stoichiometry before and after periods of forced motor stall, we measure both the number of active stator units at steady-state as a function of the load and the kinetic association and dissociation rates, by fitting the data to a reversible random sequential adsorption model. Our measurements indicate that BFM mechano-sensing relies on the dissociation rate of the stator units, which decreases with increasing load, while their association rate remains constant. This implies that the lifetime of an active stator unit assembled within the BFM increases when a higher force is applied to its anchoring point in the cell wall, providing strong evidence that a catch-bond mechanism can explain the mechano-sensitivity of the BFM.

biophysics↗

bioRxiv2017

Cell growth rate dictates the onset of glass to fluid-like transition and long time super-diffusion in an evolving cell colony

Collective migration dominates many phenomena, from cell movement in living systems to abiotic self-propelling particles. Focusing on the early stages of tumor evolution, we enunciate the principles involved in cell dynamics and highlight their implications in understanding similar behavior in seemingly unrelated soft glassy materials and possibly chemokine-induced migration of CD8+ T cells. We performed simulations of tumor invasion using a minimal three dimensional model, accounting for cell elasticity and adhesive cell-cell interactions as well as cell birth and death to establish that cell growth rate-dependent tumor expansion results in the emergence of distinct topological niches. Cells at the periphery move with higher velocity perpendicular to the tumor boundary, while motion of interior cells is slower and isotropic. The mean square displacement, {Delta}(t), of cells exhibits glassy behavior at times comparable to the cell cycle time, while exhibiting super-diffusive behavior, {Delta}(t) {approx} t ( > 1), at longer times. We derive the value of {approx} 1.33 using a field theoretic approach based on stochastic quantization. In the process we establish the universality of super-diffusion in a class of seemingly unrelated non-equilibrium systems. Super diffusion at long times arises only if there is an imbalance between cell birth and death rates. Our findings for the collective migration, which also suggests that tumor evolution occurs in a polarized manner, are in quantitative agreement with in vitro experiments. Although set in the context of tumor invasion the findings should also hold in describing collective motion in growing cells and in active systems where creation and annihilation of particles play a role.

biophysics↗

bioRxiv2017

A scheme for 3-dimensional morphological reconstruction and force inference in the early C. elegans embryo

In this study, we present novel schemes for the reconstruction of cellular morphology and the inference of forces in the early C. elegans embryo. We have developed and bench-marked a morphological reconstruction scheme that transforms live-imaging of cellular membranes into a point cloud of smoothed surface patches, which facilitates accurate estimation of membrane curvatures and the angles between membranes.\n\nAssuming an isotropic and homogeneous distribution of tensions along a membrane, we infer a pattern of forces that are 7% deviated from force balance at edges, and 10% deviated from the Young-Laplace relation at membrane faces. We have also demonstrated the stability of our scheme by sensitivity analysis of the coefficient matrices involved and the reproducibility of our image-analysis and force inference pipeline.

biophysics↗

bioRxiv2017

Cooperative mechanosensitivity and allostery of focal adhesion clusters

We analyse an Ising-like Hamiltonian describing the free energy of cell adhesion on a substrate as a lattice of 3-state mechanosensing sites involving focal adhesion kinase (FAK). We use Monte Carlo stochastic algorithm to find equilibrium configurations of these mechanosensors in two representative geometries: on a 1D ring representing the rim of a cell on flat surface, and a 2D bounded surface representing the whole area of cell contact with flat surface. The level of FAK activation depend on the pulling force applied to the individual FAKintegrin via actin-myosin contractile networks, and the details of the coupling between individual sensors in a cluster. Strong coupling is shown to make the FAK sensors experience a sharp on-off behaviour in their activation, while at low coupling the activation/autoinhibition transition occurs over a broad range of pulling force. We find that the activation/autoinhibition transition of FAK in the 2D system with strong coupling occurs with a hysteresis, the width of which depends on the rate of change of force. The effect of introducing a mediating protein (such as Src) in limited quantity to control FAK activation is explored, and visualizations of clustering in both topologies are presented. In particular the results on the bounded 2D surface indicate that clustering of active FAK occurs preferentially at the boundary, in agreement with experimental observations of focal adhesions in cells.

biophysics↗

bioRxiv2017

Small Molecules Attenuate the Interplay between Conformational Fluctuations, Early Oligomerization and Amyloidosis of Alpha Synuclein

Aggregation of alpha synuclein has strong implications in Parkinsons disease. The heterogeneity of folding/aggregation landscape and transient nature of the early intermediates result in difficulty in developing a successful therapeutic intervention. Here we used fluorescence measurements at ensemble and single molecule resolution to study how the late and early events of alpha synuclein aggregation modulate each other. The in-vitro aggregation data was complemented using measurements inside live neuroblastoma cells by employing a small molecule labeling technique. An inhibitor molecule (arginine), which delayed the late event of amyloidosis, was found to bind to the protein, shifting the early conformational fluctuations towards a compact state. In contrast, a facilitator of late aggregation (glutamate), was found to be excluded from the protein surface. The presence of glutamate was found to speed up the oligomer formation at the early stage. We found that the effects of the inhibitor and facilitator were additive and as a result they maintained a ratio at which they cancelled each others influence on different stages of alpha synuclein aggregation.

biophysics↗

bioRxiv2017

Sequence-dependent nucleosome sliding in rotation-coupled and uncoupled modes revealed by molecular simulations

While nucleosome positioning on eukaryotic genome play important roles for genetic regulation, molecular mechanisms of nucleosome positioning and sliding along DNA are not well understood. Here we investigated thermally-activated spontaneous nucleosome sliding mechanisms developing and applying a coarse-grained molecular simulation method that incorporates both long-range electrostatic and short-range hydrogen-bond interactions between histone octamer and DNA. The simulations revealed two distinct sliding modes depending on the nucleosomal DNA sequence. A uniform DNA sequence showed frequent sliding with one base pair step in a rotation-coupled manner, akin to screw-like motions. On the contrary, a strong positioning sequence, the so-called 601 sequence, exhibits rare, abrupt transitions of five and ten base pair steps without rotation. Moreover, we evaluated the importance of hydrogen bond interactions on the sliding mode, finding that strong and weak bonds favor respectively the rotation-coupled and -uncoupled sliding movements.\n\nAuthor summaryNucleosomes are fundamental units of chromatin folding consisting of double-stranded DNA wrapped [~]1.7 times around a histone octamer. By densely populating the eukaryotic genome, nucleosomes enable efficient genome compaction inside the cellular nucleus. However, the portion of DNA occupied by a nucleosome can hardly be accessed by other DNA-binding proteins, obstructing fundamental cellular processes such as DNA replication and transcription. DNA compaction and access by other proteins can simultaneously be achieved via the dynamical repositioning of nucleosomes, which can slide along the DNA sequence. In this study, we developed and used coarse-grained molecular dynamics simulations to reveal the molecular details of nucleosome sliding. We find that the sliding mode is highly dependent on the underlying DNA sequence. Specifically, a sequence with a strong nucleosome positioning signal slides via large jumps by five and ten base pairs, preserving the optimal DNA bending profile. On the other hand, uniform sequences without the positioning signal slide via a screw-like motion of DNA, one base pair at the time. These results show that sequence has a large effect not only on the formation of nucleosomes, but also on the kinetics of repositioning.

biophysics↗

bioRxiv2017

NMR studies reveal that protein dynamics critically mediate aggregation of the well-folded and very soluble E. coli S1 ribosomal protein

Unlike mammalian aging associated with many hallmarks, E. coli aging is only significantly characterized by protein aggregation, thus offering an excellent model for addressing the relationship between protein aggregation and aging. Here we characterized conformations, unfolding and dynamics of ribosomal protein S1 and its D3/D5 domains using NMR, CD and fluorescence spectroscopy. S1 is a 557-residue modular protein containing six S1 motifs. Paradoxically, while S1 is well-folded and very soluble in vitro, it was found in various lists of aggregated E. coli proteins. Our results decipher: 1) S1 has dynamic inter-domain interactions. Strikingly, S1 and its D3/D5 domains have significantly exposed hydrophobic patches characterized by irreversible unfolding. 2) Although D5 has significantly restricted backbone motion on ps-ns time scale, it has global s-ms conformational dynamics and particularly high \"global breathing\" motions. 3) D5 assumes the conserved {beta}-barrel fold but contains large hydrophobic patches at least dynamically accessible. Taken together, our study reveals that S1 could be prone to aggregation due to significant dynamics at two levels: inter-domain interactions and individual domains, which may even render buried hydrophobic patches/cores accessible for driving aggregation. This mechanism is most likely to operate in many proteins of E. coli and other organisms including human.

biophysics↗