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bioRxiv2017

Force Generation By Polymerizing Filaments Revisited: Diffusive Interaction Leads To Nonlinear Force-Number Scaling

Polymers growing against a barrier generate force and push it forward. We study here force generation by a bundle of N rigid polymers growing in parallel against a diffusing, rigid, flat barrier, resembling a bundle of microtubules. To estimate the polymerization force, the barrier is subjected to a force f acting against the direction of growth of the polymers and the mean velocity VN (f) of the filament assembly is computed. The maximum polymerization force (alias stall force) [Formula] is deduced from the condition [Formula]. This problem has been studied in the literature earlier, but two important aspects have escaped attention: (a) free diffusion of monomers is hindered by the barrier, even when it is far from the growing tips and (b) parallel filaments could interact through the monomer density field (\"diffusive coupling\"), leading to negative interference between them. In our model, both these effects are investigated in detail. A mathematical treatment based on a set of continuum Fokker-Planck equations for combined filament-wall dynamics suggests that the barrier reduces the influx of monomers to the growing polymer tip, thereby reducing the growth velocity and also the stall force, but it doesnt affect the scaling of the stall force with number, i.e., [Formula]. However, Brownian dynamics simulations show that the linear scaling holds only when the filaments are far apart; when they are arranged close together, forming a bundle, sublinear scaling of force with number appears. We argue that the nonlinear scaling could be attributed to diffusive interaction between the growing tips which becomes significant when the tips are close together. These conclusions, initially established for simple flat-faced polymers, are also found to hold true for microtubules with their characteristic hollow cylindrical geometry and rugged tip structure. In particular, simulations show conclusively that the stall force of a single microtubule is a fraction of the combined stall force of the 13 protofilaments. This result is supported by a simple analytical estimate of the force using diffusive coupling theory, and is in agreement with earlier experimental observations.

biophysics↗

bioRxiv2017

A Versatile Compressed Sensing Scheme For Faster And Less Phototoxic 3D Fluorescence Microscopy

Three-dimensional fluorescence microscopy based on Nyquist sampling of focal planes faces harsh trade-offs between acquisition time, light exposure, and signal-to-noise. We propose a 3D compressed sensing approach that uses temporal modulation of the excitation intensity during axial stage sweeping and can be adapted to fluorescence microscopes without hardware modification. We describe implementations on a lattice light sheet microscope and an epifluorescence microscope, and show that images of beads and biological samples can be reconstructed with a 5-10 fold reduction of light exposure and acquisition time. Our scheme opens a new door towards faster and less damaging 3D fluorescence microscopy.\n\nOCIS codes: (110.1758) Computational imaging; (170.2520) Fluorescence microscopy; (170.6900) Three-dimensional microscopy.

biophysics↗

bioRxiv2017

Large, long range tensile forces drive convergence during Xenopus blastopore closure and body axis elongation

Indirect evidence suggests that blastopore closure during gastrulation of anamniotes, including amphibians such as Xenopus laevis, depends on circumblastoporal convergence forces generated by the marginal zone (MZ), but direct evidence is lacking. We show that explanted MZs generate tensile convergence forces up to 1.5 N during gastrulation and over 4 N thereafter. These forces are generated by convergent thickening (CT) until the midgastrula and increasingly by convergent extension (CE) thereafter. Explants from ventralized embryos, which lack tissues expressing CE but close their blastopores, produce up to 2 N of tensile force, showing that CT alone generates forces sufficient to close the blastopore. Uniaxial tensile stress relaxation assays show stiffening of mesodermal and ectodermal tissues around the onset of neurulation, potentially enhancing long-range transmission of convergence forces. These results illuminate the mechanobiology of early vertebrate morphogenic mechanisms, aid interpretation of phenotypes, and give insight into the evolution of blastopore closure mechanisms.

biophysics↗

bioRxiv2017

Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Here we probe how the crystal packing alters microsecond dynamics, using solid-state NMR measurements and multi-microsecond MD simulations of different crystal forms of ubiquitin. In particular, NEar-Rotary-resonance Relaxation Dispersion (NERRD) experiments probe angular backbone motion, while Bloch-McConnell Relaxation Dispersion data report on fluctuations of the local electronic environment. These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. Moreover, we report small-amplitude reorientational motion of protein molecules in the crystal lattice with a [~]3-5{degrees} amplitude on a tens-of-microseconds time scale in one of the crystals, but not in others. An intriguing possibility arises that overall motion is to some extent coupled to local dynamics. Our study highlights the importance of considering the packing when analyzing dynamics of crystalline proteins.

biophysics↗

bioRxiv2017

Comparison Of NMR And Crystal Structures Of Membrane Proteins And Computational Refinement To Improve Model Quality

Membrane proteins are challenging to study and restraints for structure determination are typically sparse or of low resolution because the membrane environment that surrounds them leads to a variety of experimental challenges. When membrane protein structures are determined by different techniques in different environments, a natural question is \"which structure is most biologically relevant?\" Towards answering this question, we compiled a dataset of membrane proteins with known structures determined by both solution NMR and X-ray crystallography. By investigating differences between the structures, we found that RMSDs between crystal and NMR structures are below 5 [A] in the membrane region, NMR ensembles have a higher convergence in the membrane region, crystal structures typically have a straighter transmembrane region, have higher stereo-chemical correctness, and are more tightly packed. After quantifying these differences, we used high-resolution refinement of the NMR structures to mitigate them, which paves the way for identifying and improving the structural quality of membrane proteins.

biophysics↗

bioRxiv2017

The Unique Protein-To-Protein Carotenoid Transfer Mechanism

List of abbreviations\n\nAbstractOrange Carotenoid Protein (OCP) is known to be an effector and regulator of cyanobacterial photoprotection. This 35 kDa water-soluble protein provides specific environment for keto-carotenoids, the excitation of which induced by the absorption of blue-green light causes dramatic but fully reversible rearrangements of the OCP structure, including carotenoid translocation and separation of C- and N-terminal domains upon transition from the basic orange to photoactivated red OCP form. While recent studies significantly improved our understanding of the OCP photocycle and interaction with phycobilisomes and the fluorescence recovery protein, the mechanism of OCP assembly remains unclear. Apparently, this process requires targeted delivery and incorporation of a highly hydrophobic carotenoid molecule into the water-soluble apoprotein of OCP. Recently, we introduced a novel carotenoid carrier protein, COCP, which consists of dimerized C-domain(s) of OCP and can combine with the isolated N-domain to form transient OCP-like species. Here, we demonstrate that in vitro COCP efficiently transfers otherwise tightly bound carotenoid to the full-length OCP apoprotein, resulting in formation of the photoactive OCP from completely photoinactive species. We accurately analyze peculiarities of this carotenoid transfer process which, to the best of our knowledge, seems unique, previously uncharacterized protein-to-protein carotenoid transfer process. We hypothesize that a similar OCP assembly can occur in vivo, substantiating specific roles of the COCP carotenoid carrier in cyanobacterial photoprotection.

biophysics↗

bioRxiv2017

Characterization Of The Stoichiometry Of The Complex Formed By Staphylococcal LukSF And Human C5aR Receptor In Living Cells

Staphylococcus aureus Panton Valentine Leukocidin (PVL) is a pore-forming toxin comprising protein subunits LukS and LukF. Binding of LukS to human C5a receptor (hC5aR) on leukocytes induces secondary binding of LukF and assembly of lytic complexes. Previous analysis suggests that PVL consists of 4-plus-4 LukS/LukF subunits but the exact stoichiometry between LukS, LukF and hC5aR is not yet known. In this study we determine the stoichiometry and spatiotemporal dynamics of functional LukS/LukF-hC5aR complexes in living eukaryotic cells. By using rapid total internal reflection fluorescence (TIRF) and single-molecule photobleaching analysis we found that tetrameric LukS-hC5aR complexes are formed within a cluster of receptors. Upon binding to hC5aR each LukS subunit binds LukF leading to lytic pore formation and simultaneous dissociation of receptors from the complex. Our findings corroborate a hetero-octamer model but provide a new view on the kinetics of crucial virulence factor assembly on integrated host cell membrane receptors.

biophysics↗

bioRxiv2017

A model for autonomous and non-autonomous effects of the Hippo pathway in Drosophila

While significant progress has been made toward understanding morphogen-mediated patterning in development from both the experimental and the theoretical side, the control of size and shape of tissues and organs is poorly understood. Both involve adjustment of the scale of gene expression to the size of the system, but how growth and patterning are coupled to produce scale invariance and how molecular-level information is translated into organ- and organism-level functioning is one of the most difficult problems in biology. The Hippo pathway, which controls cell proliferation and apoptosis in Drosophila and mammalian cells, contains a core kinase mechanism that affects control of the cell cycle and growth. Studies involving over- and under-expression of components in the morphogen and Hippo pathways in Drosophila reveal conditions that lead to over- or undergrowth. Herein we develop a mathematical model that incorporates the current understanding of the Hippo signal transduction network and which can explain qualitatively both the observations on whole-disc manipulations and the results arising from mutant clones. We find that a number of non-intuitive experimental results can be explained by subtle changes in the balances between inputs to the Hippo pathway. Since signal transduction and growth control pathways are highly conserved across species, much of what is learned about Drosophila applies in higher organisms, and may have direct relevance to tumor dynamics in mammalian systems.

biophysics↗

bioRxiv2017

A multi-model approach to assessing local and global cryo-EM map quality

There does not currently exist a standardized indicator of how well a cryo-EM-derived model represents the density from which it was generated. We present a straightforward methodology that utilizes freely available tools to generate a suite of independent models and to evaluate their convergence in an EM density. These analyses provide both a quantitative and qualitative assessment of the precision of the models and their representation of the density, respectively, while concurrently providing a platform for assessing both global and local EM map quality. We further use standardized datasets to provide an expected model-model agreement criterion for EM maps reported to be at 5 [A] resolution or better. Associating multiple atomic models with a deposited EM map provides a rapid and accessible reporter of convergence, a strong indicator of highly resolved molecular detail, and is an important step toward an FSC-independent assessment of map and model quality.

biophysics↗

bioRxiv2017

Matrix Production In Bacillus subtilis Biofilms Is Localized To A Propagating Front

Bacterial biofilms are surface attached microbial communities encased in self-produced extracellular polymeric substances (EPS). Development of a mature biofilm must require coordinating cell differentiation and multicellular activity at scales much larger than the single microbial unit. Here we demonstrate that during development of Bacillus subtilis biofilms, EPS matrix production is localized to a front propagating at the periphery. We show that within the front, cells switch off matrix production and transition to sporulation after a set time delay of[~] 100 min. Correlation analyses of fluctuations in fluorescence reporter activity reveals that the front emerges from a pair of gene expression waves of matrix production and sporulation. The expression waves travel across cells that are immobilized in the biofilm matrix, in contrast to active cell migration or horizontal colony spreading. A single length scale and time scale couples the spatiotemporal propagation of both fronts throughout development, with the front displacement obeying a t1/2 scaling law. As a result, gene expression patterns within the advancing fronts collapse to self-similar expression profiles. Our results indicate that development of bacterial biofilms may be governed by universal wave-like dynamics localized to a self-similar front.

biophysics↗

bioRxiv2017

Optimal Information Transfer In Enzymatic Networks: A Field Theoretic Formulation

Signaling in enzymatic networks is typically triggered by environmental fluctuations, resulting in a series of stochastic chemical reactions, leading to corruption of the signal by noise. For example, information flow is initiated by binding of extracellular ligands to receptors, which is transmitted through a cascade involving kinase-phosphatase stochastic chemical reactions. For a class of such networks, we develop a general field-theoretic approach in order to calculate the error in signal transmission as a function of an appropriate control variable. Application of the theory to a simple push-pull network, a module in the kinase-phosphatase cascade, recovers the exact results for error in signal transmission previously obtained using umbral calculus (Phys. Rev. X., 4, 041017 (2014)). We illustrate the generality of the theory by studying the minimal errors in noise reduction in a reaction cascade with two connected push-pull modules. Such a cascade behaves as an effective three-species network with a pseudo intermediate. In this case, optimal information transfer, resulting in the smallest square of the error between the input and output, occurs with a time delay, which is given by the inverse of the decay rate of the pseudo intermediate. Surprisingly, in these examples the minimum error computed using simulations that take non-linearities and discrete nature of molecules into account coincides with the predictions of a linear theory. In contrast, there are substantial deviations between simulations and predictions of the linear theory in error in signal propagation in an enzymatic push-pull network for a certain range of parameters. Inclusion of second order perturbative corrections shows that differences between simulations and theoretical predictions are minimized. Our study establishes that a field theoretic formulation of stochastic biological signaling offers a systematic way to understand error propagation in networks of arbitrary complexity.

biophysics↗

bioRxiv2017

Gradients Of Rac1 Nanoclusters Support Spatial Patterns Of Rac1 Signaling

The dynamics of the cytoskeleton and cell shape relies on the coordinated activation of RhoGTPase molecular switches. Among them, Rac1 participates to the orchestration in space and time of actin branching and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single molecule imaging and super-resolution microscopy, we reveal an additional supramolecular organization of Rac1. We find that, similarly to H-Ras, Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble due to the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs, GAPs, downstream effectors, and possibly other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Using optogenetics and micropatterning tools, we find that activation of Rac1 leads to its immobilization in nanoclusters and that the local level of Rac1 activity matches the local density of nanoclusters. Altogether, our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms. This finding implies that graded distributions of nanoclusters might encode spatial information.\n\nSignificance statementThe plasma membrane of eukaryotic cells is a highly organized surface where hundreds of incoming signals are transduced to the intracellular space. How cells encode faithfully this myriad of signals is a fundamental question. Here we show that Rac1, a critical membrane-bound protein involved in the regulation of cytoskeletal dynamics, forms small aggregates together with other regulating proteins. These supramolecular assemblies, called nanoclusters, are the \"quantal\" units of signaling. By increasing the local concentration, nanoclusters set thresholds for downstream signaling and ensure the fidelity of information transduction. We found that Rac1 nanoclusters are distributed as spatial gradients matching the patterns of Rac1 activity. We propose that cells can encode positional information through distributed signaling quanta, hereby ensuring spatial fidelity.

biophysics↗

bioRxiv2017

Structure-based analysis of CysZ-mediated cellular uptake of sulfate

Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. Current understanding of the cellular delivery of SO42- at the molecular level is limited however. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and uptake experiments in cells and proteoliposomes, and single-channel conductance measurements, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ properties differ markedly from those of known transporters and ion channels. The structures display a hitherto unknown fold with dual topology, assembling in CysZ from Pseudomonas denitrificans as a trimer of antiparallel dimers in the membrane. CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.

biophysics↗

bioRxiv2017

Quantifying the efficacy of first aid treatments for burn injuries using mathematical modelling and in vivo porcine experiments

First aid treatment of burn injuries reduces scarring and improves healing. Here, we quantify the efficacy of various first aid treatments by using a mathematical model to describe a suite of experimental data from a series of in vivo porcine experiments. We study a series of consistent burn injuries that are subject to first aid treatments that vary in both the temperature and duration of the first aid treatment. Calibrating the mathematical model to the experimental data provides estimates of the in vivo thermal diffusivity, the rate at which thermal energy is lost to the blood (perfusion), and the heat transfer coefficient controlling the loss of thermal energy at the interface of the fat and muscle layers. A limitation of working with in vivo animal experiments is the difficulty of resolving spatial variations in temperature across the tissues. Here, we use the solution of the calibrated mathematical model to predict and visualise the temperature distribution across the thickness of the tissue during the creation of the burn injury and the application of various first aid treatments. Using this information we propose, and report values for, a novel measure of the potential for tissue damage. This measure quantifies two important aspects that are thought to be related to thermal injury: (i) the volume of tissue that rises above the threshold temperature associated with the accumulation of tissue damage; and, (ii) the duration of time that the tissue remains above this threshold temperature. We conclude by discussing the clinical relevance of our findings.

biophysics↗

bioRxiv2017

Structural Basis Of STAT2 Recognition By IRF9 Reveals Molecular Insights Into ISGF3 Function

Cytokine signalling is mediated by the activation of distinct sets of structurally homologous JAK and STAT signalling molecules, which control nuclear gene expression and cell fate. A significant expansion in the gene regulatory repertoire controlled by JAK/STAT signalling has arisen by the selective interaction of STATs with IRF transcription factors. Type I interferons (IFN), the major antiviral cytokines, trigger the formation of the ISGF3 complex containing STAT1, STAT2 and IRF9. ISGF3 regulates the expression of IFN-stimulated genes (ISGs). ISGF3 assembly depends on selective interaction between IRF9, through its IRF-association domain (IAD), with the coiled-coil domain (CCD) of STAT2. Here, we report the crystal structures of the IRF9-IAD alone and in a complex with STAT2-CCD. Despite similarity in the overall structure among respective paralogs, the surface features of the IRF9-IAD and STAT2- CCD have diverged to enable specific interaction between these family members, thus enabling ISGF3 formation and expression of ISGs.

biophysics↗

bioRxiv2017

Modeling Of Biomechanics And Biorheology Of Red Blood Cells In Type-2 Diabetes Mellitus

Erythrocytes in patients with type-2 diabetes mellitus (T2DM) are associated with reduced cell deformability and elevated blood viscosity, which contribute to impaired blood flow and other pathophysiological aspects of diabetes related vascular complications. In this study, by using a two-component red blood cell (RBC) model and systematic parameter variation, we perform detailed computational simulations to probe the alteration of the biomechanical, rheological and dynamic behavior of T2DM RBCs in response to morphological change and membrane stiffening. First, we examine the elastic response of T2DM RBCs subject to static tensile forcing and their viscoelastic relaxation response upon release of the stretching force. Second, we investigate the membrane fluctuations of T2DM RBCs and explore the effect of cell shape on the fluctuation amplitudes. Third, we subject the T2DM RBCs to shear flow and probe the effects of cell shape and effective membrane viscosity on their tank-treading movement. In addition, we model the cell dynamic behavior in a microfluidic channel with constriction and quantify the biorheological properties of individual T2DM RBCs. Finally, we simulate T2DM RBC suspensions under shear and compare the predicted viscosity with experimental measurements. Taken together these simulation results and their comparison with currently available experimental data are helpful in identifying a specific parametric model the first of its kind that best describes the main hallmarks of T2DM RBCs, which can be used in future simulation studies of hematologic complications of T2DM patients.

biophysics↗

bioRxiv2017

Emerging Electrical Properties of Graphene incorporated Photosynthetic Biofilms

We report emergence of a new electrical material by growing photosynthetic biofilm on a Dirac material, graphene. The material showed new conducting as well as semiconducting properties. Frequency dependent capacitive spectra further indicated presence of electrical isosbestic points(at 0.8 and 9MHz), implying two state dieletric transitions at critical frequencies. A notable reult was a Schottky diode like behavior in the IV curve. Voltage dependent conductance with conductance peaks near the Schottky diode threshold was observed. We obtained facilitated growth of photosynthetic biofilm in presence of graphene. Lastly higher bacterial metabolism i was seen in the biofilm in contact with graphene as compared to its normal growth condition. For this zero band gap Dirac material this can only be interpreted as coupling of the electron transport chain of the bacterial biofilm and the graphene electron cloud.

biophysics↗

bioRxiv2017

High-Density Super-Resolution Microscopy With An Incoherent Light Source And A Conventional Epifluorescence Microscope Setup

We report that single-molecule superresolution microscopy can be achieved with a conventional epifluorescence microscope setup and a Mercury arc lamp. The configuration termed as Omnipresent Localisation Microscope (OLM), is an extension of Single Molecule Localisation Microscopy (SMLM) techniques and allows single molecules to be switched on and off (blinking), detected and localised. The use of a short burst of deep blue excitation can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, for the first time, we demonstrate that STED and OLM can be performed on the same biological sample using a simple imaging buffer. It is hoped that such a correlative imaging will provide a basis for a further enhanced resolution.\n\nScope of the findingsDespite ten years of development, superresolution microscopy is still limited to relatively few microscopy and optics groups. This is mainly due to the significant cost of the superresolution microscopes which require high-quality lasers, high NA objective lens, a very sensitive camera, a highly precise microscope stage, and a complex post-acquisition data reconstruction and analysis. We present results that demonstrate the possibility to obtain nanoscale resolution images using a conventional microscope and an incoherent light source. We show an easyto-follow protocol that every biologist can implement in the laboratory. We hope that this finding will help any scientist to generate high-density super-resolution images even with limited budget. Lastly, the new photophysical observations reported here will pave the way for more in-depth investigations on excitation, photobleaching and photoactivation of a fluorophore.

biophysics↗