PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.17.246082v1?rss=1

Authors: Cochet-Escartin, O., Demircigil, M., Hirose, S., Allais, B., Calvez, V., Funamoto, K., Rieu, J.-P.

Abstract:

It is well known that eukaryotic cells can sense oxygen (O2) and adapt their metabolism accordingly. It is less known that they can also move towards regions of higher oxygen level (aerotaxis). Using a self-generated hypoxic assay, we show that the social amoeba Dictyostelium discoideum displays a spectacular aerotactic behavior. When a cell colony is covered by a coverglass, cells quickly consume the available O2 and the ones close to the periphery move directionally outward forming a dense ring keeping a constant speed and density. To confirm that O2 is the main molecular player in this seemingly collective process, we combined two technological developments, porphyrin based O2 sensing films and microfluidic O2 gradient generators. We showed that Dictyostelium cells exhibit aerotactic and aerokinetic (increased speed at low O2) response in an extremely low range of O2 concentration (0-1.5%) indicative of a very efficient detection mechanism. The various cell behaviors under self-generated or imposed O2 gradients were modeled with a very satisfactory quantitative agreement using an in silico cellular Potts model built on experimental observations. This computational model was complemented with a parsimonious 'Go or Grow' partial differential equation (PDE) model. In both models, we found that the collective migration of a dense ring can be explained by the interplay between cell division and the modulation of aerotaxis, without the need for cell-cell communication. Explicit wave solutions of the PDE model also informed about the relative contributions of division and directed motion on the collective speed.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.17.253567v1?rss=1

Authors: Schneider, M. C., Telschow, R., Mercier, G., Lopez-Martinez, M., Scherzer, O., Schütz, G.

Abstract:

Single molecule localization microscopy (SMLM) has enormous potential for resolving subcellular structures below the diffraction limit of light microscopy: Localization precision in the low digit nanometer regime has been shown to be achievable. In order to record localization microscopy data, however, sample fixation is inevitable to prevent molecular motion during the rather long recording times of minutes up to hours. Eventually, it turns out that preservation of the sample's ultrastructure during fixation becomes the limiting factor. We propose here a workflow for data analysis, which is based on SMLM performed at cryogenic temperatures. Since molecular dipoles of the fluorophores are fixed at low temperatures, such an approach offers the possibility to use the orientation of the dipole as an additional information for image analysis. In particular, assignment of localizations to individual dye molecules becomes possible with high reliability. We quantitatively characterized the new approach based on the analysis of simulated oligomeric structures. Side lengths can be determined with a relative error of less than 1% for tetramers with a nominal side length of 5 nm, even if the assumed localization precision for single molecules is more than 2 nm.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.17.244137v1?rss=1

Authors: Kahil, K., Varsano, N., Sorrentino, A., Pereiro, E., Rez, P., Weiner, S., Addadi, L.

Abstract:

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy (cryo-SXM) we imaged intra-cellular calcium-containing particles in the PMCs and acquired Ca-L2,3 X-ray absorption near edge spectra (XANES) of these Ca-particles. Using the pre-peak/main peak (L2'/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-particles with order parameters, L2'/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1-15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all water, is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.17.253609v1?rss=1

Authors: Solowiej-Wedderburn, J., Dunlop, C.

Abstract:

Cell mechanotransduction, in which cells sense and respond to the physical properties of their micro-environments, is proving fundamental to understanding cellular behaviours across biology. Tissue stiffness (Youngs modulus) is typically regarded as the key control parameter and bioengineered gels with defined mechanical properties have become an essential part of the toolkit for interrogating mechanotransduction. We here, however, show using a mechanical cell model that the effective substrate stiffness experienced by a cell depends not just on the engineered mechanical properties of the substrate but critically also on the particular arrangement of adhesions between cell and substrate. In particular, we find that cells with different adhesion patterns can experience two different gel stiffnesses as equivalent and will generate the same mean cell deformations. For small adhesive patches, which mimic experimentally observed focal adhesions, we demonstrate that the observed dynamics of adhesion growth and elongation can be explained by energy considerations. Significantly we show different focal adhesions dynamics for soft and stiff substrates with focal adhesion growth not preferred on soft substrates consistent with reported dynamics. Equally, fewer and larger adhesions are predicted to be preferred over more and smaller, an effect enhanced by random spot placing with the simulations predicting qualitatively realistic cell shapes in this case. The model is based on a continuum elasticity description of the cell and substrate system, with an active stress component capturing cellular contractility. This work demonstrates the necessity of considering the whole cell-substrate system, including the patterning of adhesion, when investigating cell stiffness sensing, with implications for mechanotransductive control in biophysics and tissue engineering.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.15.252437v1?rss=1

Authors: Malladi, S. K., Singh, R., Pandey, S., Gayathri, S., Kanjo, K., Ahmed, S., Khan, M. S., Kalita, P., Girish, N., Upadhyaya, A., Reddy, P., Pramanick, I., Bhasin, M., Mani, S., Bhattacharyya, S., Joseph, J., Thankamani, K., Raj, V. S., Dutta, S., Singh, R., Nadig, G., Varadarajan, R.

Abstract:

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel, nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 200mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100{degrees}C when lyophilized, and upto 70{degrees}C in solution. In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.16.253013v1?rss=1

Authors: Vo, Q. N., Mahinthichaichan, P., Shen, J., Ellis, C. R.

Abstract:

The opioid crisis has escalated during the COVID-19 pandemic. More than half of the overdose-related deaths are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, crystal structures of mOR complexed with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like synthetic opioids remains lacking. Exploiting the X-ray structure of mOR bound to a morphinan ligand and several state-of-the-art simulation techniques, including weighted ensemble and continuous constant pH molecular dynamics, we elucidated the detailed binding mechanism of fentanyl with mOR. Surprisingly, in addition to the orthosteric site common to morphinan opiates, fentanyl can move deeper and bind mOR through hydrogen bonding with a conserved histidine H297, which has been shown to modulate mOR's ligand affinity and pH dependence in mutagenesis experiments, but its precise role remains unclear. Intriguingly, the secondary binding mode is only accessible when H297 adopts a neutral HID tautomer. Alternative binding modes and involvement of tautomer states may represent general mechanisms in G protein-coupled receptor (GPCR)-ligand recognition. Our work provides a starting point for understanding mOR activation by fentanyl analogs that are emerging at a rapid pace and assisting the design of safer analgesics to combat the opioid crisis. Current protein simulation studies employ standard protonation and tautomer states; our work demonstrates the need to move beyond the practice to advance our understanding of protein-ligand recognition.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.15.252643v1?rss=1

Authors: Kumar, A., Tagad, A., Patwari, G. N.

Abstract:

Ribose containing double-stranded nucleic acids exhibit helical structure, whereas sug-ar modified (xeno) nucleic acids may exhibit different structural features. The structural landscape of four stereo variants of furanosal nucleic acids and their C2' deoxy counter-parts, explored with molecular dynamics simulations, suggest that the configuration at the C3' position plays a pivotal role in determining the helicity. The C3' stereocentre acts as toggle-switch for the helix to ladder structural transformation by changing the nature of intra-strand interactions resulting in the optimal helices for ribose containing double-stranded nucleic acids. Interestingly, lack of chirality at the C2' position results in better quality helices than inversion of stereochemistry relative to ribose. The etiolo-gy of furanosal-RNA over other furanoses can be hypothesized based on the helical structure, which can effectively be exploited by the biological machinery.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.16.253070v1?rss=1

Authors: Palovcak, E., Asarnow, D., Campbell, M. G., Yu, Z., Cheng, Y.

Abstract:

In cryogenic electron microscopy (cryo-EM) of radiation-sensitive biological samples, both the signal-to-noise ratio (SNR) and the contrast of images are critically important in the image processing pipeline. Classic methods improve low-frequency image contrast experimentally, by imaging with high defocus, or computationally, by applying various types of low-pass filter. These contrast improvements typically come at the expense of high-frequency SNR, which is suppressed by high-defocus imaging and removed by low pass filtration. Here, we demonstrate that a convolutional neural network (CNN) denoising algorithm can be used to significantly enhance SNR and generate contrast in cryo-EM images. We provide a quantitative evaluation of bias introduced by the denoising procedure and its influences on image processing and three-dimensional reconstructions. Our study suggests that besides enhancing the visual contrast of cryo-EM images, the enhanced SNR of denoised images may facilitate better outcomes in the other parts of the image processing pipeline, such as classification and 3D alignment. Overall, our results provide a ground of using denoising CNNs in the cryo-EM image processing pipeline.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.15.250811v1?rss=1

Authors: Da Costa, A. D. S., Subiah, R., Oh, S. J., Jeong, H. T., Na, J.-I., Park, K., Shin, J. H., Choi, I.-S.

Abstract:

How stromal cells fill voids in wounded tissue remains one of the most fundamental questions in regenerative medicine. Fibroblasts are known to fill voids by depositing extracellular matrix (ECM) proteins while migrating towards the wound site; however, their ability to adopt an epithelial-like purse-string behaviour remains unexplored. Here, we fabricated an artificial wound with a deep void space to investigate fibroblasts' behaviour during gap closure. We found that fibroblasts can form a free-standing bridge on deep microvoids and consequently close the void through the purse-string contraction, which was previously believed to be exclusively an epithelial wound closure mechanism. The results also revealed that the fibroblast gap closure in our fabricated 3D artificial wound depends on myosin II-mediated contractility and intercellular adherent junctions. Our study reveals that stromal cells can gain the structural features of epithelial cells, namely, intercellular contractile rings, to fulfil their functions under the specific microenvironmental conditions of tissue repair. Furthermore, fibroblasts can close artificial wounds with gap widths up to 300 m, approximately twice as large as the critical epithelial gap closure size on non-adherent substrates. Fibroblasts exhibited a zip-up gap closure mechanism with a geometrical size effect. These findings reveal a new mechanism for gap closure by stromal cells during wound healing and pave a way to groundbreaking therapeutic strategies for tissue repair.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.08.15.252346v1?rss=1

Authors: Ruff, K. M., Dar, F., Pappu, R. V.

Abstract:

Biomolecular condensates are thought to form via phase transitions of multivalent macromolecules. Components of condensates have been classified as scaffolds and clients. In this classification, scaffolds drive condensate formation whereas clients partition into condensates. However, non-scaffold molecules can be ligands that modulate the phase behavior of scaffolds via preferential binding across phase boundaries. Here, we present computational results that explain how preferential binding of ligands to scaffold molecules in different phases can impact phase separation and the compositions of dense phases. We show that phase separation is destabilized by monovalent ligands. In contrast, multivalent ligands, depending on their mode of binding, can stabilize phase separation by serving as crosslinkers that network scaffold molecules. We also find that concentrations of scaffolds within dense phases are generally diluted by ligands. This arises because ligands destabilize condensates by preferentially binding to scaffolds in the dilute phase or because ligands have to be accommodated within dense phases when they bind preferentially to scaffolds in the dense phase. Importantly, we show that partition coefficients, which are routinely used for compositional profiling of condensates, say nothing about the regulatory impact of ligands on the phase behavior of scaffolds. Therefore, instead of measuring partition coefficients it becomes imperative to measure how ligands impact threshold concentrations of scaffolds. These measurements, performed as a function of ligand concentration, will help with understanding the regulation of condensates and enable the design of molecules that impact condensate formation or dissolution.

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