PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.15.252247v1?rss=1

Authors: Motiwala, Z., Sandholu, A. S., Sengupta, D., Kulkarni, K.

Abstract:

Ras superfamily GTPases are molecular switches which regulate critical cellular processes. Extensive structural and computational studies on this G protein family have tried to establish a general framework for their switching mechanism. The current understanding of the mechanism is that two loops, Switch I and Switch II, undergo conformational changes upon GTP binding and hydrolysis, which results in alteration of their functional state. However, because of variation in the extent of conformational changes seen across the members of the Ras superfamily, there is no generic modus operandi defining their switching mechanism, in terms of loop conformation. Here, we have developed a novel method employing wavelet transformation to dissect the structures of these molecular switches to explore indices that defines the unified principle of working. Our analysis shows that the structural coupling between the Switch I and Switch II regions is manifested in terms of wavelet coherence phases. The resultant phase pertaining to these regions serve as a functional identity of the GTPases. The coupling defined in terms of wavelet coherence phases is conserved across the Ras superfamily. In oncogenic mutants of the GTPases the phase coupling gets disentangled, this perhaps provides an alternative explanation for their aberrant function. Although similar observations were made using MD simulations, there was no structural parameter to define the coupling, as delineated here. Furthermore, the technique reported here is computationally inexpensive and can provide significant functional insights on the GTPases by analyzing as few as two structures.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.14.232587v1?rss=1

Authors: Yan, R., Chen, K., Xu, K.

Abstract:

Diffusion properties notably determine the behavior of biomembranes. Here we report the concurrent nanoscale fine-mapping of membrane topography, diffusivity, and packing order in live mammalian cells through a synergy of single-molecule and super-resolution methods. By identifying a bright, lipophilic fluorescence turn-on probe that enables sustained single-molecule imaging of cellular membranes under stroboscopic excitation, we accumulate the positions and transient displacements of >10^6 probe molecules to achieve super-resolution topography and diffusivity mapping. We thus determine a trend that the membrane diffusivity drops with increased lipid packing order when comparing the endoplasmic reticulum (ER) membrane, plasma membrane, and nanodomains induced by cholera toxin B. Utilizing our nanoscale mapping capability, we further unveil reduced diffusivity in the ER membrane at ER-plasma membrane contact sites. By next integrating spectrally resolved single-molecule imaging, we show this localized diffusion slowdown is not due to altered lipid packing order, but may instead be attributed to local protein crowding. Our integrated multidimensional single-molecule approach thus unveils and differentiates between nanoscale diffusional heterogeneities of different origins in live-cell membranes.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.13.250233v1?rss=1

Authors: Caceres-Delpiano, J., Wang, L.-P., Essex, J. W.

Abstract:

Atomistic models provide a detailed representation of molecular systems, but are sometimes inadequate for simulations of large systems over long timescales. Coarse-grained models enable accelerated simulations by reducing the number of degrees of freedom, at the cost of reduced accuracy. New optimisation processes to parameterise these models could improve their quality and range of applicability. We present an automated approach for the optimisation of coarse-grained force fields, by reproducing free energy data derived from atomistic molecular simulations. To illustrate the approach, we implemented hydration free energy gradients as a new target for force field optimisation in ForceBalance and applied it successfully to optimise the un-charged side-chains and the protein backbone in the SIRAH protein coarse-grain force field. The optimised parameters closely reproduced hydration free energies of atomistic models and gave improved agreement with experiment.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.14.250506v1?rss=1

Authors: Pardon, G., Castillo, E. A., Pruitt, B.

Abstract:

Traction Force Microscopy (TFM) has become a well-established technique to assay the biophysical force produced by cells cultured on soft substrates of controlled stiffness. However, experimental conditions as well as computational implementations can have a large impact on the analysis results accuracy and reproducibility. While this can be alleviated using appropriate controls and a rigorous analytical approach, the comparison of results across studies remains difficult and there is a need for validation and benchmarking tools. To validate the accuracy of and compare various computational TFM analysis algorithms, we developed a virtual in silico model of a cell contracting on a soft substrate of controlled stiffness. The model utilizes user-defined parameters for the cell dimensions as well as for the strength and spatial distribution of a contraction dipole to calculate the deformation that would result on a soft substrate due to the cell contraction. The deformation is computed using the forward analytical stress-strain tensor calculation in the Fourier space. The resulting displacement field is used to apply, using image processing, a deformation on a real or simulated image of fluorescent microspheres embedded into a soft hydrogel, which is normally obtain experimentally by TFM imaging. The deformation field and resulting image then serve as input in the PIV and TFM analysis. The model also enables to create movies of a dynamic cell contraction, such as that of a cardiomyocyte, to validate the time accuracy of the TFM analysis after application of image processing algorithm, such as denoising. Our tool therefore addresses the need for validation and standardization of TFM analytical algorithms and its experimental implementations.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.13.247304v1?rss=1

Authors: Miller, C. M., Korkmazhan, E., Dunn, A. R.

Abstract:

Dynamic remodeling of the actin cytoskeleton allows cells to migrate, change shape, and exert mechanical forces on their surroundings. How the complex dynamical behavior of the cytoskeleton arises from the interactions of its molecular components remains incompletely understood. Tracking the movement of individual actin filaments in living cells can in principle provide a powerful means of addressing this question. However, single-molecule fluorescence imaging measurements that could provide this information are limited by low signal-to-noise ratios, with the result that the localization errors for individual fluorophore fiducials attached to filamentous (F)-actin are comparable to the distances traveled by actin filaments between measurements. In this study we tracked the movement F-actin labeled with single-molecule densities of the fluorogenic label SiR-actin in primary fibroblasts and endothelial cells. We then used a Bayesian statistical approach to estimate true, underlying actin filament velocity distributions from the tracks of individual actin-associated fluorophores along with quantified localization uncertainties. This analysis approach is broadly applicable to inferring statistical pairwise distance distributions arising from noisy point localization measurements such as occur in superresolution microscopy. We found that F-actin velocity distributions were better described by a statistical jump process, in which filaments exist in mechanical equilibria punctuated by abrupt, jump-like movements, than by models incorporating combinations of diffusive motion and drift. A model with exponentially distributed time- and length-scales for filament jumps recapitulated F-actin velocity distributions measured for the cell cortex, integrin-based adhesions, and actin stress fibers, indicating that a common physical model can potentially describe F-actin dynamics in a variety of cellular contexts.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.13.249466v1?rss=1

Authors: Avidan, C., Day, S. W., Holzman, R.

Abstract:

Ubiquitous constraints derived from the muscle's structure limit the power capacity of fast contracting muscles. Correspondingly, organisms evolved elastic elements that store energy which, when released, can be used to rapidly accelerate appendages. Such latch-mediated spring actuation (LaMSA) systems comprise of a single elastic element and are used to actuate a single mass. Here we reveal a dual LaMSA system in seahorses, in which two elastic elements actuate two masses: the head as they rapidly swing it towards the prey, and the water mass sucked into the mouth to prevent the prey from escaping. This power-amplified system enhances the speeds of both head rotation and suction flows by x10 compared to similarly-sized fish. Furthermore, the dual system provides temporal coordination between head rotation and suction flows, a novel function for LaMSA. These findings extend the known function, capacity and design of LaMSA systems.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.13.249144v1?rss=1

Authors: Tilunaite, A., Ladd, D., Hunt, H., Soeller, C., Roderick, H., Crampin, E. J., Rajagopal, V.

Abstract:

Calcium plays critical roles in cardiac cells, coupling electrical excitation to mechanical contraction with each heartbeat, while simultaneously mediating biochemical signals that regulate cell growth. While ryanodine receptors (RyRs) are fundamental to generation of elementary calcium release events (sparks) and global calcium elevations that underlie excitation-contraction coupling (ECC), calcium release via inositol 1,4,5-trisphosphate receptors (IP3Rs) is also reported in cardiomyocytes. IP3R calcium release modifies ECC as well as contributing to downstream regulation of hypertrophic gene expression. Recent studies suggest that proximal localisation of IP3Rs with RyRs contributes to their ability to modify Ca2+ handling during ECC. Here we aim to determine the mechanism by which IP3Rs modify Ca2+ handling in cardiomyocytes. We develop a mathematical model incorporating the stochastic behaviour of receptor opening that allows for the parametric tuning of the system to reveal the impact of IP3Rs on spark activation. By testing multiple spark initiation mechanisms, we find that Ca2+ release via IP3Rs result in increased propensity for spark initiation within the cardiac dyad. Our simulations suggest that opening of IP3Rs elevates Ca2+ within the dyad, which increase the probability of spark initiation. Finally, we find that while increasing the number of IP3Rs increases the probability of spark formation, it has little effect on spark amplitude, duration, or overall shape. Our study therefore suggests that IP3R play a critical role in modulating Ca2+ signaling for excitation contraction coupling.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.12.248633v1?rss=1

Authors: Pauszek, R., Lamichhane, R., Rajkarnikar Singh, A., Millar, D.

Abstract:

Replication and repair of genomic DNA requires the action of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA polymerase I (Pol I) from E. coli, a key enzyme involved in lagging-strand replication and base excision repair. Pol I contains active sites for template-directed DNA polymerization and 5' flap processing in separate domains. We show that a DNA substrate can spontaneously transfer between polymerase (pol) and 5' nuclease (5' nuc) domains during a single encounter with Pol I. Additionally, we show that the flexibly tethered 5' nuc domain adopts different positions within Pol I-DNA complexes, depending on the nature of the DNA substrate. Our results reveal the structural dynamics that underlie functional coordination in Pol I and are likely relevant to other multi-functional DNA polymerases.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.14.250951v1?rss=1

Authors: Shetty, R. M., Brady, S. R., Rothemund, P. W., Hariadi, R. F., Gopinath, A.

Abstract:

Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations. Until recently, creating such grids which combine the unique advantages of microarrays and single-molecule experiments (SMEs) has been particularly challenging due to the mismatch between the size of these molecules and the resolution of top-down fabrication techniques. DNA Origami Placement (DOP) combines two powerful techniques to address this issue: (i) DNA origami, which provides a 100-nm self-assembled template for single-molecule organization with 5 nm resolution, and (ii) top-down lithography, which patterns these DNA nanostructures, transforming them into functional nanodevices via large-scale integration with arbitrary substrates. Presently, this technique relies on state-of-the-art infrastructure and highly-trained personnel, making it prohibitively expensive for researchers. Here, we introduce a bench-top technique to create meso-to-macro-scale DNA origami nanoarrays using self-assembled colloidal nanoparticles, thereby circumventing the need for top-down fabrication. We report a maximum yield of 74%, two-fold higher than the statistical limit of 37% imposed on non-specific molecular loading alternatives. Furthermore, we provide a proof-of-principle for the ability of this nanoarray platform to transform traditionally low-throughput, stochastic, single-molecule assays into high-throughput, deterministic ones, without compromising data quality. Our approach has the potential to democratize single-molecule nanoarrays and demonstrates their utility as a tool for biophysical assays and diagnostics.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.14.251363v1?rss=1

Authors: Shepherd, J., Lecinski, S., Wragg, J., Shashkova, S., MacDonald, C., Leake, M. C.

Abstract:

The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress.

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