PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.27.223941v1?rss=1

Authors: Brown, P. T., Kruithoff, R., Seedorf, G. J., Shepherd, D. P.

Abstract:

We demonstrate structured illumination microscopy (SIM) at three wavelengths using a digital mirror device (DMD), an approach which has not previously been realized due to the DMD's blazed grating effect. Previous approaches to SIM using a DMD have used one color of coherent light or multiple colors via incoherent projection. To address this challenge, we develop a forward model of DMD diffraction, a forward model of coherent pattern projection, and identify an experimentally feasible configuration for three common fluorophore wavelengths. Based on these results, we constructed a custom DMD SIM and created an open-source software suite to simulate, calibrate, execute, and reconstruct 2D linear SIM data. We validate this approach by demonstrating resolution enhancement for known calibration samples and fixed cells.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.28.224741v1?rss=1

Authors: Paraschiv, A., Lagny, T., Coudrier, E., Bassereau, P., Saric, A.

Abstract:

The cell membrane is an inhomogeneous system composed of phospholipids, sterols and proteins that can be directly attached to underlying cytoskeleton. The linkers between the membrane and the cytoskeleton are believed to have a profound effect on the mechanical properties of the cell membrane and its ability to reshape. Here we investigate the role of membrane-cortex linkers on the extrusion of membrane tubes using computer simulations and experiments. In simulations we find that the force for tube extrusion has a non-linear dependence on the density of membrane-cortex attachments: at a wide range of low and intermediate densities of linkers the force is not significantly influenced by the presence of membrane linking proteins and resembles that of the bare membrane. For large concentrations of linkers however the force substantially increases compared to the bare membrane. In both cases the linkers provided membrane tubes with increased stability against coalescence. We then pulled tubes from HEK cells using optical-tweezers for varying expression levels of the membrane-cortex attachment protein Ezrin. In line with simulations, we observed that overexpression of Ezrin led to an increased extrusion force, while Ezrin depletion had negligible effect on the force. Our results shed new light on the importance of local effects in membrane reshaping at the nanoscopic scales.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.24.219774v1?rss=1

Authors: Lee, A. J., Hugonnet, H., Park, W., Park, Y.

Abstract:

The wound healing assay provides essential information about collective cell migration and cell-to-cell interactions. It is a simple, effective, and widely used tool for observing the effect of numerous chemical treatments on wound healing speed. To perform and analyze a wound healing assay, various imaging techniques have been utilized. However, image acquisition and analysis are often limited in two-dimensional space or require the use of exogenous labeling agents. Here, we present a method for imaging large-scale wound healing assays in a label-free and volumetric manner using optical diffraction tomography (ODT). We performed quantitative high-resolution three-dimensional (3D) analysis of cell migration over a long period without difficulties such as photobleaching or phototoxicity. ODT enables the reconstruction of the refractive index (RI) tomogram of unlabeled cells, which provides both structural and biochemical information about the individual cell at subcellular resolution. Stitching multiple RI tomograms enables long-term (24 h) and large field-of-view imaging (> 800 x 400 m2) with a lateral resolution of 110 nm. We demonstrated the thickness changes of leading cells and studied the effects of cytochalasin D. The 3D RI tomogram also revealed increased RI values in leading cells compared to lagging cells, suggesting the formation of a highly concentrated subcellular structure.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.27.221150v1?rss=1

Authors: Burra, G., Maina, M. B., Serpell, L. C., Thakur, A. K.

Abstract:

An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY serves as an ideal model for structural understanding of amyloid assembly and kinetics. In this study, we used a reproducible solubilisation protocol that allows the generation of homogenous monomeric solution of GNNQQNY to understand the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequences follow nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the size and efficiency of nucleation was found to be inversely related to the reaction temperature. The generated nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T positivity, Congo red binding and {beta}-sheet rich structures displaying a characteristic cross-{beta} diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers exerted no significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous predictions based on theoretical and molecular dynamics simulations. These findings will provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic systems associated with many systemic and neurodegenerative diseases.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.24.219782v1?rss=1

Authors: Eberhardt, P., Slavov, C., Sörmann, J., Bamann, C., Braun, M., Wachtveitl, J.

Abstract:

Here we applied target analysis to a temperature dependent flash photolysis dataset of the light-driven sodium ion pump Krokinobacter rhodopsin 2 (KR2) at sodium pumping conditions. With an increase in temperature from 10 - 40 {degrees}C, the overall photocycle duration was accelerated by a factor of six, while single transitions like the L to M transition increased by a factor of 40. Using kinetic modeling with the Eyring constraint as well as spectral corrections on the datasets the spectral position as well as the equilibria of the different photointermediates could be resolved. The results provide further insight into KR2s photocycle and energetics.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.27.219618v1?rss=1

Authors: Mulla, Y., Avellaneda, M. J., Roland, A., Baldauf, L., Tans, S. J., Koenderink, G.

Abstract:

Molecular catch bonds are ubiquitous in biology and well-studied in the context of leukocyte extravasion, cellular mechanosensing, and urinary tract infection. Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this remarkable ability enables cells to increase their adhesion in fast fluid flows, and hence provides "strength-on-demand". Recently, cytoskeletal crosslinkers have been discovered that also display catch bonding. It has been suggested that they strengthen cells, following the strength-on-demand paradigm. However, catch bonds tend to be weaker compared to regular (slip) bonds because they have cryptic binding sites that are often inactive. Therefore, the role of catch bonding in the cytoskeleton remains unclear. Here we reconstitute cytoskeletal actin networks to show that catch bonds render them both stronger and more deformable than slip bonds, even though the bonds themselves are weaker. We develop a model to show that weak binding allows the catch bonds to mitigate crack initiation by moving from low- to high-tension areas in response to mechanical loading. By contrast, slip bonds remain trapped in stress-free areas. We therefore propose that the mechanism of catch bonding is typified by dissociation-on-demand rather than strength-on-demand. Dissociation-on-demand can explain how both cytolinkers and adhesins exploit continuous redistribution to combine mechanical strength with the adaptability required for movement and proliferation. Our findings provide a mechanistic understanding of diseases where catch bonding is compromised such as kidney focal segmental glomerulosclerosis, caused by the -actinin-4 mutant studied here. Moreover, catch bonds provide a route towards creating life-like materials that combine strength with deformability.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.27.222802v1?rss=1

Authors: Giubertoni, G., Burla, F., Bakker, H. J., Koenderink, G.

Abstract:

Many biopolymer hydrogels are environmentally responsive because they are held together by physical associations that depend on pH and temperature. Here we investigate how the pH and temperature response of the rheology of hyaluronan hydrogels is connected to the underlying molecular interactions. Hyaluronan is an essential structural biopolymer in the human body with many applications in biomedicine. Using two-dimensional infrared (2DIR) spectroscopy, we show that hyaluronan chains become connected by hydrogen bonds when the pH is changed from 7.0 to 2.5, and that the bond density at pH 2.5 is independent of temperature. Temperature-dependent rheology measurements show that due to this hydrogen bonding the stress relaxation at pH 2.5 is strongly slowed down in comparison to pH 7.0, consistent with the sticky reptation model of associative polymers. From the flow activation energy we conclude that each polymer is crosslinked by multiple (5-15) hydrogen bonds to others, causing slow macroscopic stress relaxation, despite the short time scale of breaking and reformation of each individual hydrogen bond. Our findings can aid the design of stimuli-responsive hydrogels with tailored viscoelastic properties for biomedical applications.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.27.223727v1?rss=1

Authors: Sacco, M. D., Ma, C., Lagarias, P., Gao, A., Townsend, J. A., Meng, X., Dube, P., Zhang, X., Hu, Y., Kitamura, N., Hurst, B., Tarbet, B., Marty, M. T., Kolocouris, A., Xiang, Y., Chen, Y., Wang, J.

Abstract:

The main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mpro inhibitors have a gamma-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogues of GC-376, one of the most potent Mpro inhibitors in vitro. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket of Mpro can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1 prime pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1 prime pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three GC-376 analogues UAWJ246, UAWJ247, and UAWJ248 provide insight to the sidechain preference of the S1 prime, S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the alpha-ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mpro inhibitors as SARS-CoV-2 antivirals.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.24.219188v1?rss=1

Authors: Ries, J.

Abstract:

Reconstruction of superresolved images from the raw data of blinking fluorophores in single-molecule localization microscopy (SMLM) requires extensive data analysis. Current software solutions often lack functionality, continuous development and cannot easily be extended with own algorithms. Here we release as open source a comprehensive Superresolution Microscopy Analysis Platform (SMAP) for SMLM. Its modular architecture makes it easy for anyone with basic programming experience to add own plugins, limiting development effort to implementing algorithms. It has a freely configurable graphical user interface (GUI) and currently contains more than 200 plugins spanning all steps of data analysis from single molecule fitting to post-processing, rendering and advanced quantitative analyses. SMAP is a powerful, ready-to-use software package for all steps of SMLM data analysis, that enables anyone to use state-of-the art algorithms and complex analysis workflows and that provides a versatile platform for developers to share and publish new SMLM algorithms.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.26.221770v1?rss=1

Authors: Cao, E., Zhao, Y., Zhou, M., Shen, J., Wang, Q.

Abstract:

Secondary active cation-chloride cotransporters (CCCs) catalyze electroneutral symport of Cl- with Na+ and/or K+ across membranes1,2. CCCs are fundamental in cell volume homeostasis, transepithelia ion movement, maintenance of intracellular Cl- concentration, and inhibitory synaptic transmission3-6. K+-Cl- cotransport 1 (KCC1) was first characterized in red blood cells and later in many other cell types as a crucial player in regulatory volume decrease in defense against cell swelling upon hypotonic challenges7,8. Here we present two cryo-EM structures of human KCC1: one captured in an inward-open state and another arrested in an outward-open state by a small molecule inhibitor. KCC1 can surprisingly adopt two distinct dimeric architectures via homotypic association of different protein domains and conversion between these two forms of dimers may entail dynamic formation and rupture of two interdigitating regulatory cytoplasmic domains. The inhibitor wedges into and forces open an extracellular ion permeation path and arrests KCC1 in an outward-open conformation. Concomitantly, the outward-open conformation involves inward movement of the transmembrane helix 8 and occlusion of the intracellular exit by a conserved short helix within the intracellular loop 1. Our structures provide a blueprint for understanding the mechanisms of CCC transporters and their inhibition by small molecule compounds.

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