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Response-Time Behaviors Of Intercellular Communication Network Motifs

Cell-to-cell communication networks have critical roles in diverse organismal processes, such as coordinating tissue development or immune cell response. However, compared to intracellular signal transduction networks, the function and engineering principles of cell-to-cell communication networks are far less understood. Here, we study cell-to-cell communication networks using a framework that models the input-to-output relationship of intracellular signal transduction networks with a single function--the response-time distribution. We identify a prototypic response-time distribution--the gamma distribution--arising in both experimental data sets and mathematical models of signal-transduction pathways. We find that simple cell-to-cell communication circuits can generate bimodal response-time distributions, and can control synchronization and delay of cell-population responses independently. We apply our modeling approach to explain otherwise puzzling data on cytokine secretion onset times in T cells. Our approach can be used to predict communication network structure using experimentally accessible input-to-output measurements and without detailed knowledge of intermediate steps.

biophysics

Gaussian Curvature Directs The Distribution Of Spontaneous Curvature On Bilayer Membrane Necks

Formation of membrane necks is crucial for fission and fusion in lipid bilayers. In this work, we seek to answer the following fundamental question: what is the relationship between protein-induced spontaneous mean curvature and the Gaussian curvature at a membrane neck? Using an augmented Helfrich model for lipid bilayers to include membrane-protein interaction, we solve the shape equation on catenoids to find the field of spontaneous curvature that satisfies mechanical equilibrium of membrane necks. In this case, the shape equation reduces to a variable coefficient Helmholtz equation for spontaneous curvature, where the source term is proportional to the Gaussian curvature. We show how this latter quantity is responsible for non-uniform distribution of spontaneous curvature in minimal surfaces. We then explore the energetics of catenoids with different spontaneous curvature boundary conditions and geometric asymmetries to show how heterogeneities in spontaneous curvature distribution can couple with Gaussian curvature to result in membrane necks of different geometries.

biophysics

The Condensin Complex Is A Mechanochemical Motor That Translocates Along DNA

One Sentence SummarySingle-molecule imaging reveals that eukaryotic condensin is a highly processive DNA-translocating motor complex.\n\nAbstractCondensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. Here, we use single-molecule imaging to demonstrate that Saccharomyces cerevisiae condensin is a molecular motor capable of ATP hydrolysis-dependent translocation along double-stranded DNA. Condensins translocation activity is rapid and highly processive, with individual complexes traveling an average distance of [≥]10 kilobases at a velocity of [~]60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its [~]50-nanometer coiled-coil subunits, suggestive of a translocation mechanism that is distinct from any reported DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.

biophysics

Conformational Dynamics In TRPV1 Channels Reported By An Encoded Coumarin Amino Acid

Transient Receptor Potential Vanilloid (TRPV1) channels support the detection and integration of nociceptive input. Currently available functional and structural data suggest that that TRPV1 channels have two potential gates within their cation selective permeation pathway: a barrier formed by a bundle crossing at the intracellular entrance and a second constriction created by the ion selectivity filter. To describe conformational changes associated with channel gating within the pore, the fluorescent non-canonical amino acid (f- ncAA) coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter. TRPV1 channels expressing coumarin at either site displayed normal voltage- and agonist-dependent gating. Next, total internal reflection microscopy (TIRF) was performed to enable ultra-rapid, millisecond imaging of the conformational dynamics in single TRPV1 channels in live cells. Here, the data obtained from channels expressed in human derived cells show that optical fluctuations, photon counts, and variance of noise analysis from Y671 coumarin encoded in TRPV1 tetramers correlates closely with channel activation by capsaicin, thus providing an direct optical marker of channel activation at the selectivity filter. In companion molecular dynamics simulations, Y671 displays alternating solvent exposure between the closed and open states, giving support to the optical data. These calculations further suggest a direct involvement of Y671 in controlling the relative position of the pore helix and its role in supporting ionic conductance at the TRPV1 selectivity filter.

biophysics

Defocus and magnification dependent variation of TEM image astigmatism

Daily alignment of the microscope is a prerequisite to reaching optimal illumination lens and imaging lens conditions for high resolution imaging in cryo-EM. In contrast to the dramatic progress in automated image acquisition and post-image processing techniques, less attention has been paid to the improvement of microscope alignment before data collection. In this study, we have employed our recently published tool, s2stigmator, to study how image astigmatism varies with the imaging conditions (e.g. defocus, magnification). We have found that the large change of defocus/magnification between visual correction of astigmatism and subsequent data collection tasks, or even during data collection will inevitably result in undesirable residual astigmatism in the final images. Furthermore, the dependence of astigmatism on the imaging conditions varies significantly from time to time, so that it cannot be reliably compensated by pre-calibration of the microscope. These findings have essentially invalidated a basic assumption of current cryo-EM imaging strategies that assumes invariant astigmatism for different defocuses/magnifications used in the microscope alignment stage and the final data acquisition stage. Based on these findings, we recommend the same magnification and the median defocus of the intended defocus range for final data collection are used in the objective lens astigmatism correction task during microscope alignment and in the focus mode of the iterative low-dose imaging. It is also desirable to develop a fast, accurate method that can perform dynamic correction of the astigmatism for different intended defocuses during automated imaging.

biophysics

The AWSEM-Amylometer: Predicting Amyloid Propensity And Fibril Topology Using An Optimized Folding Landscape Model

Amyloids are fibrillar protein aggregates with simple repeated structural motifs in their cores, usually {beta}-strands but sometimes -helices. Identifying the amyloid-prone regions within protein sequences is important both for understanding the mechanisms of amyloid-associated diseases and for understanding functional amyloids. Based on the crystal structures of seven cross-{beta} amyloidogenic peptides with different topologies and one recently solved cross- fiber structure, we have developed a computational approach for identifying amyloidogenic segments in protein sequences using the Associative memory, Water mediated, Structure and Energy Model. The AWSEM-Amylometer performs favorably in comparison with other predictors in predicting aggregation-prone sequences in multiple datasets. The method also predicts the specific topologies (the relative arrangement of {beta}-strands in the core) of the amyloid fibrils well. An important advantage of the AWSEM-Amylometer over other existing methods is its direct connection with an efficient, optimized protein folding simulation model, AWSEM. This connection allows one to combine efficient and accurate search of protein sequences for amyloidogenic segments with the detailed study of the thermodynamic and kinetic roles that these segments play in folding and aggregation in the context of the entire protein sequence. We present new simulation results that highlight the free energy landscapes of peptides that can take on multiple fibril topologies. We also demonstrate how the Amylometer methodology can be straightforwardly extended to the study of functional amyloids that have the recently discovered cross- fibril architecture.

biophysics

Poor man's two photon imaging: Scanning Laser Upconversion Microscopy

Two photon microscopy is one of the most powerful techniques for optical images. Its deep penetration, low scattering and mainly its sectioning power allows exquisite control on all three axes of 3D samples, both for imaging and actuation. Unfortunately, this technique involves very expensive femtosecond lasers. Upconverting Nanoparticles display multiphoton absorption at low instantaneous power, which can be achieved by means of cheap laser diodes. Scanning Laser Upconversion Microscopy offers the deep penetration and low scattering of NIR excitation together with the sectioning power of multiphoton techniques for under US$ 1000.

biophysics

A Liquid Chromatography-Mass Spectrometry Method For Screening Disulfide Tethering Fragments

We report the refinement of a high-throughput, liquid-chromatography/mass spectrometry (LC/MS)-based screening method for the identification of covalent small-molecule binders to proteins. Using a custom library of 1600 disulfide-capped fragments targeting surface cysteine residues, we optimize sample preparation, chromatography, and ionization conditions to maximize the reliability and flexibility of the approach. Data collection at a rate of 90 seconds per sample balances speed and reliability for sustained screening over multiple, diverse projects run over a 24-month period. The method is applicable to protein targets of various classes and a range of molecular masses. Data are processed in a custom pipeline that calculates a % bound value for each compound and detects false-positives by calculating significance of detected masses ( signal significance). An example pipeline has been made available through Biovias ScienceCloud Protocol Exchange. Data collection and analysis methods for the screening of covalent adducts of intact proteins are now fast enough to screen the largest covalent compound libraries in 1-2 days.

biophysics

A Combined Use Of Intravoxel Incoherent Motion MRI Parameters Can Differentiate Early Stage Hepatitis-B Fibrotic Livers From Healthy Livers

This study investigated a combined use of IVIM parameters Dslow (D), PF (f) and Dfast (D*) for liver fibrosis evaluation. 16 healthy volunteers (F0) and 33 hepatitis-b patients (stage F1= 15, stage F2-4 = 18) were included. With a 1.5-T MR scanner and respiration-gating, IVIM diffusion weighted imaging was acquired using a single-shot echo-planar imaging sequence with ten b-values of 10, 20, 40, 60, 80, 100, 150, 200, 400, and 800 s/mm2. Signal measurement was performed on right liver parenchyma. With a 3-dimensional tool, Dslow, PF, and Dfast values were placed along the x-axis, y-axis, and z-axis, and a plane was defined to separate healthy volunteers from patients. 3-dimensional tool demonstrated healthy volunteers and all patients with liver fibrosis could be separated. Classification and Regression Tree showed a combination of PF (PF < 12.55%), Dslow (Dslow < 1.152 x10-3 mm2/s) and Dfast (Dfast <13.36 x10-3 mm2/s) could differentiate healthy subjects and all fibrotic livers (F1-F4) with an area under the curve of logistic regression (AUC) of 0.986. The AUC for differentiation of healthy livers vs. F2-4 livers was 1. PF offered the best diagnostic value, followed by Dslow; however, all three parameters of PF, Dslow, and Dfast contributed to liver fibrosis detection.

biophysics

Cryo-EM Of A Marseilleviridae Virus Particle Reveals A Large Internal Microassembly

Nucleocytoplasmic large DNA viruses (NCLDVs) blur the line between viruses and cells. Melbournevirus (MelV, fam. Marseilleviridae) belongs to a new family of NCLDVs. Here we present an electron cryo-microscopy structure of the MelV particle, with the largest known triangulation number (T=309) for a virus. The 230-nm particle is constructed by 3080 pseudo-hexagonal capsomers and encloses a membrane bilayer. Its most distinct feature is a large dense body (LDB) consistently found in all particles. Electron cryo-tomography of 147 particles showed that the LDB is located preferentially in proximity to the bilayer. The LDB is 30 nm in size and its density matches that of a genome/protein complex. More than 58 proteins are associated with the purified particle, including histone-like proteins, putative membrane proteins and capsid proteins. The observed intricate structural organization reinforces the genetic complexity of MelV, setting it apart from other viruses, and suggests an evolutionary link with cellular organisms.

biophysics

Control Of AMPA Receptor Activity By The Extracellular Loops Of Auxiliary Proteins

At synapses throughout the mammalian brain, AMPA receptors form complexes with auxiliary proteins, including TARPs. However, how TARPs modulate AMPA receptor gating remains poorly understood. We built structural models of TARP-AMPA receptor complexes for TARPs {gamma}2 and {gamma}8, combining recent structural studies and de novo structure predictions. These models, combined with peptide binding assays, provide evidence for multiple interactions between GluA2 and variable extracellular loops of TARPs. Substitutions and deletions of these loops had surprisingly rich effects on the kinetics of glutamate-activated currents, without any effect on assembly. Critically, by altering the two interacting loops of {gamma}2 and {gamma}8, we could entirely remove all allosteric modulation of GluA2, without affecting formation of AMPA receptor-TARP complexes. Likewise, substitutions in the linker domains of GluA2 completely removed any effect of Y2 on receptor kinetics, indicating a dominant role for this previously overlooked site proximal to the AMPA receptor channel gate.

biophysics

Cooperative Accumulation Of Dynein-Dynactin At Microtubule Minus-Ends Drives Microtubule Network Reorganization

SummaryCytoplasmic dynein-1 (dynein) is minus-end directed motor protein that transports cargo over long distances and organizes microtubules (MTs) during critical cellular processes such as mitotic spindle assembly. How dynein motor activity is harnessed for these diverse functions remains unknown. Here, we have uncovered a mechanism for how processive dynein-dynactin complexes drive MT-MT sliding, reorganization, and focusing, activities required for mitotic spindle assembly. We find that motors cooperatively accumulate, in limited numbers, at MT minus-ends. Minus-end accumulations drive MT-MT sliding, independent of MT orientation, and this activity always results in the clustering of MT minus-ends. At a mesoscale level, activated dynein-dynactin drives the formation and coalescence of MT asters. Macroscopically, dynein-dynactin activity leads to bulk contraction of millimeter-scale MT networks, demonstrating that minus-end accumulations produce network scale contractile stresses. Our data provides a model for how localized dynein activity is harnessed by cells to produce contractile stresses within the mitotic spindle.\n\nHighlightsO_LIProcessive dynein-dynactin complexes cooperatively form stable accumulations at MT minus-ends.\nC_LIO_LIMinus-end accumulations of motors slide MTs without orientation bias, leading to minus-end focusing.\nC_LIO_LIMinus-end accumulations of motors organize dynamic MTs into asters.\nC_LIO_LIMinus-end accumulations of motors drive bulk contractions of large-scale MT networks.\nC_LI

biophysics

Molecular Dynamics Simulations Of Membrane Deformation Induced By The Amphiphilic Helices Of Epsin, Sar1p And Arf1

The N-terminal amphiphilic helices of proteins Epsin, Sar1p and Arf1 play a critical role in initiating membrane deformation. We present here the study of the interactions of these amphiphilic helices with the lipid membranes by combining the all-atom and coarse-grained simulations. In the all-atom simulations, we find that the amphiphilic helices of Epsin and Sar1p have a shallower insertion depth into the membrane compared to the amphiphilic helix of Arf1, but remarkably, the amphiphilic helices of Epsin and Sar1p induce higher asymmetry in the lipid packing between the two monolayers of the membrane. The insertion depth of amphiphilic helix into the membrane is determined not only by the overall hydrophobicity but also by the specific distribution of polar and non-polar residues along the helix. To directly compare their ability of deforming the membrane, we further apply coarse-grained simulations to investigate the membranes deformation under the insertion of multiple helices. Importantly, it is found that the amphiphilic helices of Epsin and Sar1p generate a larger membrane curvature than that of Arf1, in accord with the experimental results qualitatively. These findings enhance our understanding of the molecular mechanism of the protein-driven membrane remodeling.

biophysics

Disruption Of Ankyrin B And Caveolin-1 Interaction Sites Alters Na+,K+-ATPase Lateral Diffusion In HEK293 Cell Plasma Membranes

The Na+,K+-ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na+,K+-ATPase -subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2 (FHM2), Alternating Hemiplegia of Childhood (AHC), Rapid Dystonia Parkinsonism (RDP) or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to e.g. reduced protein stability, reduced protein expression or defective plasma membrane targeting. Na+,K+-ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutational disruption of established interaction motifs of the Na+,K+-ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na+,K+-ATPase constructs in the plasma membrane of living HEK293T cells by fluorescence correlation spectroscopy (FCS) as well as fluorescence recovery after photobleaching (FRAP) or photoswitching (FRAS) and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na+,K+-ATPase mutations and provide information about the interaction of Na+,K+-ATPase 2-isoform with cellular matrix proteins, the cytoskeleton or other membrane protein complexes.

biophysics

Filament Rigidity And Connectivity Tune The Deformation Modes Of Active Biopolymer Networks

Molecular motors embedded within collections of actin and microtubule filaments underlie the dynamic behaviors of cytoskeletal assemblies. Understanding the physics of such motor-filament materials is critical to developing a physical model of the cytoskeleton and the design of biomimetic active materials. Here, we demonstrate through experiments and simulations that the rigidity and connectivity of filaments in active biopolymer networks regulates the anisotropy and the length scale of the underlying deformations, yielding materials with varying contractility. Semi-flexible filaments that can be compressed and bent by motor stresses undergo deformations that are predominantly biaxial. By contrast, rigid filament bundles contract via actomyosin sliding deformations that are predominantly uniaxial. Networks dominated by filament buckling are robustly contractile under a wide range of connectivities, while networks dominated by actomyosin sliding can be tuned from contractile to extensile through reduced connectivity via cross-linking. These results identify physical parameters that control the forces generated within motor-filament arrays, and provide insight into the self-organization and mechanics of cytoskeletal assemblies.

biophysics

A kinetically-driven free exchange mechanism of EmrE antiport sacrifices coupling efficiency in favor of promiscuity

EmrE is a small multidrug resistance transporter found in E. coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: Not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a new kinetically-driven free exchange model for EmrE antiport that is consistent with these results and recapitulates {Delta}pH-driven concentrative drug uptake. Our results suggest that EmrE sacrifices coupling efficiency for initial transport speed and multidrug specificity.\n\nSIGNIFICANCEEmrE facilitates E. coli multidrug resistance by coupling drug efflux to proton import. This antiport mechanism has been thought to occur via a pure exchange model which achieves coupled antiport by restricting when the single binding pocket can alternate access between opposite sides of the membrane. We test this model using NMR titrations and transport assays and find it cannot account for EmrE antiport activity. We propose a new kinetically-driven free exchange model of antiport with fewer restrictions that better accounts for the highly promiscuous nature of EmrE drug efflux. This model expands our understanding of coupled antiport and has implications for transporter design and drug development.

biophysics

Achieving Better Than 3 A Resolution By Single Particle Cryo-EM At 200 keV

Technical and methodological advances in single-particle cryo-electron microscopy (cryo-EM) have expanded the technique into a resolution regime that was previously only attainable by X-ray crystallography. Although single-particle cryo-EM has proven to be a useful technique for determining the structures of biomedically relevant molecules at near-atomic resolution, nearly 98% of the structures resolved to better than 4 [A] resolution have been determined using 300 keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain cryo-EM reconstructions of macromolecular complexes at a range of sizes to better than 3 [A] resolution using a 200 keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules, features previously thought only to be resolvable using TEMs operating at 300 keV.

biophysics

Detailed Analyses Of Stall Force Generation In Mycoplasma mobile Gliding

Mycoplasma mobile is a bacterium that uses a unique mechanism to glide on solid surfaces at a velocity of up to 4.5 {micro}m/s. Its gliding machinery comprises hundreds of units that generate the force for gliding based on the energy derived from ATP; the units catch and pull on sialylated oligosaccharides fixed to solid surfaces. In the present study, we measured the stall force of wild-type and mutant strains of M. mobile carrying a bead manipulated using optical tweezers. The strains that had been enhanced for binding exhibited weaker stall forces than the wild-type strain, indicating that stall force is related to force generation rather than to binding. The stall force of the wild-type strain decreased linearly from 113 to 19 pN following the addition of 0-0.5 mM free sialyllactose (a sialylated oligosaccharide), with a decrease in the number of working units. Following the addition of 0.5 mM sialyllactose, the cells carrying a bead loaded using optical tweezers exhibited stepwise movements with force increments. The force increments ranged from 1 to 2 pN. Considering the 70-nm step size, this small unit force may be explained by the large gear ratio involved in the M. mobile gliding machinery.\n\nSIGNIFICANCEMycoplasma is a genus of bacteria that parasitizes animals. Dozens of Mycoplasma species glide over the tissues of their hosts during infection. The gliding machinery of Mycoplasma mobile, the fastest species, includes intracellular motors and hundreds of legs on the cell surface. In the present study, we precisely measured force generation using a highly focused laser beam arrangement (referred to as optical tweezers) under various conditions. The measurements obtained in this study suggest that the rapid gliding exhibited by M. mobile arises from the large gear ratio of its gliding machinery.

biophysics