Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 54,955 bioRxiv papers from 253,602 authors.
Most downloaded bioRxiv papers, since beginning of last month
in category biophysics
2,197 results found. For more information, click each entry to expand.
181 downloads biophysics
Mammalian genomes are folded into Topologically Associating Domains (TADs), consisting of cell-type specific chromatin loops anchored by CTCF and cohesin. Since CTCF and cohesin are expressed ubiquitously, how cell-type specific CTCF-mediated loops are formed poses a paradox. Here we show RNase-sensitive CTCF self-association in vitro and that an RNA-binding region (RBR) mediates CTCF clustering in vivo. Intriguingly, deleting the RBR abolishes or impairs almost half of all chromatin loops in mouse embryonic stem cells. Disrupted loop formation correlates with abrogated clustering and diminished chromatin binding of the RBR mutant CTCF protein, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least 2 classes: RBR-independent and RBR-dependent loops. We suggest that evidence for distinct classes of RBR-dependent loops may provide a mechanism for establishing cell-specific CTCF loops regulated by RNAs and other RBR partners.
178 downloads biophysics
The mammalian cell nucleus displays a remarkable spatial segregation of active euchromatic from inactive heterochromatic genomic regions. In conventional nuclei, euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery. In contrast, rod photoreceptors in nocturnal mammals have inverted nuclei, with a dense heterochromatic core and a thin euchromatic outer shell. This inverted architecture likely converts rod nuclei into microlenses to facilitate nocturnal vision, and may relate to the absence of particular proteins that tether heterochromatin to the lamina. However, both the mechanism of inversion and the role of interactions between different types of chromatin and the lamina in nuclear organization remain unknown. To elucidate this mechanism we performed Hi-C and microscopy on cells with inverted nuclei and their conventional counterparts. Strikingly, despite the inversion evident in microscopy, both types of nuclei display similar Hi-C maps. To resolve this paradox we developed a polymer model of chromosomes and found a universal mechanism that reconciles Hi-C and microscopy for both inverted and conventional nuclei. Based solely on attraction between heterochromatic regions, this mechanism is sufficient to drive phase separation of euchromatin and heterochromatin and faithfully reproduces the 3D organization of inverted nuclei. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. To further test our models, we eliminated lamina interactions in models of conventional nuclei and found that this triggers a spontaneous process of inversion that qualitatively reproduces the pathway of morphological changes during nuclear inversion in vivo. Together, our experiments and modeling suggest that interactions among heterochromatic regions are central to phase separation of the active and inactive genome in inverted and conventional nuclei, while interactions with the lamina are essential for building the conventional architecture from these segregated phases. Ultimately our data suggest that an inverted organization constitutes the default state of nuclear architecture.
177 downloads biophysics
Plasma membranes dynamically respond to external cues and changing environment. Quantitative measurements of these adaptations can elucidate the mechanism that cells exploit to survive, adapt and function. However, cell-based assays are affected by active processes while measurements on synthetic models suffer from compositional limitations. Here, as a model system we employ giant plasma membrane vesicles (GPMVs), which largely preserve the plasma membrane lipidome and proteome. From analysis of fluorescence emission and lifetime of environment-sensitive dyes, and membrane shape fluctuations, we investigate how plasma membrane order, viscosity and bending rigidity are affected by different stimuli such as cell seeding density in three different cell models. Our studies reveal that bending rigidity of plasma membranes vary with lipid order and microviscosity in a highly correlated fashion. Thus, readouts from polarity- and viscosity-sensitive probes represent a promising indicator of membrane mechanical properties. Quantitative analysis of the data allows for comparison to synthetic lipid membranes as plasma membrane mimetics.
173 downloads biophysics
Carolina O. Matos, Yulli M. Passos, Mariana J Amaral, Bruno Macedo, Matheus Tempone, Ohanna C. L. Bezerra, Milton O. Moraes, Marcius S. Almeida, Gerald Weber, Sotiris Missailidis, Jerson L. Silva, Anderson S. Pinheiro, Yraima Cordeiro
Structural conversion of cellular prion protein (PrPC) into scrapie PrP (PrPSc) and subsequent aggregation are key events for the onset of Transmissible Spongiform Encephalopathies (TSEs). Experimental evidences support the role of nucleic acids (NAs) in assisting the protein conversion process. Here, we used the SELEX methodology to identify two 25-mer DNA aptamers against the globular domain of recombinant murine PrP (rPrP90-231), namely A1 and A2. High-affinity binding of A1 and A2 to rPrP was verified by ITC. Aptamers structure was characterized by theoretical predictions, CD, NMR and SAXS, revealing that A1 adopts a hairpin conformation. Aptamer binding caused dynamic aggregation of rPrP90-231, resulting from the ability of rPrP90-231 to undergo liquid-liquid phase separation (LLPS). While free rPrP90-231 phase separated into large droplets, aptamer binding increased the amount but reduced the size of the condensates. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced transition to an ordered state, suggestive of amyloid formation on the surface of the droplets. Our results describe for the first time PrP:NA interaction leading to LLPS and modulation of this effect depending on NA structure and binding stoichiometry, shedding light on the role of NAs in PrP misfolding and TSEs.
173 downloads biophysics
Chromatin nanoscale architecture in live cells can be studied by Forster Resonance Energy Transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor-donor ratio rather than distance. Here we show that a FRET value independent of the acceptor-donor ratio can be obtained by Fluorescence Lifetime Imaging (FLIM) detection of FRET combined with a normalization of the FRET level to a pixel-wise estimation of the acceptor-donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that acceptor-donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser-induced micro-irradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.
170 downloads biophysics
Genomic DNA is highly compacted in the nucleus of eukaryotic cells as a nucleoprotein assembly called chromatin. The basic unit of chromatin is the nucleosome, where ~146 base pair increments of the genome are wrapped and compacted around the core histone proteins. Further genomic organization and compaction occur through higher order assembly of nucleosomes. This organization regulates many nuclear processes, and is controlled in part by histone post-transtranslational modifications and chromatin-binding proteins. Mechanisms that regulate the assembly and compaction of the genome remain unclear. Here we show that in the presence of physiologic concentrations of mono- and divalent salts, histone tail-driven interactions drive liquid-liquid phase separation (LLPS) of nucleosome arrays, resulting in substantial condensation. Phase separation of nucleosomal arrays is inhibited by histone acetylation, whereas histone H1 promotes phase separation, further compaction, and decreased dynamics within droplets, mirroring the relationship between these modulators and the accessibility of the genome in cells. These results indicate that under physiologically relevant conditions, LLPS is an intrinsic behavior of the chromatin polymer, and suggest a model in which the condensed phase reflects a genomic 'ground state' that can produce chromatin organization and compaction in vivo. The dynamic nature of this state could enable known modulators of chromatin structure, such as post-translational modifications and chromatin binding proteins, to act upon it and consequently control nuclear processes such as transcription and DNA repair. Our data suggest an important role for LLPS of chromatin in the organization of the eukaryotic genome.
169 downloads biophysics
Jervis Vermal Thevathasan, Maurice Kahnwald, Konstanty Cieslinski, Philipp Hoess, Sudheer Kumar Peneti, Manuel Reitberger, Daniel Heid, Krishna Chaitanya Kasuba, Sarah Janice Hoerner, Yiming Li, Yu-Le Wu, Markus Mund, Ulf Matti, Pedro Matos Pereira, Ricardo Henriques, Bianca Nijmeijer-Winter, Moritz Kueblbeck, Vilma Jimenez Sabinina, Jan Ellenberg, Jonas Ries
Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures, to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting. These cell lines will enable the broad community to assess the quality of their microscopes and labels and to perform quantitative, absolute measurements.
167 downloads biophysics
The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimens is currently largely uncoupled from subsequent data evaluation, correction and processing. Therefore, the acquisition strategy is difficult to optimize during data collection, often leading to suboptimal microscope usage and disappointing results. Here we provide Warp, a software for real-time evaluation, correction, and processing of cryo-EM data during their acquisition. Warp evaluates and monitors key parameters for each recorded micrograph or tomographic tilt series in real time. Warp also rapidly corrects micrographs for global and local motion, and estimates the local defocus with the use of novel algorithms. The software further includes a deep learning-based particle picking algorithm that rivals human accuracy to make the pre-processing pipeline truly automated. The output from Warp can be directly fed into established tools for particle classification and 3D image reconstruction. In a benchmarking study we show that Warp automatically processed a published cryo-EM data set for influenza virus hemagglutinin, leading to an improvement of the nominal resolution from 3.9 Å to 3.2 Å. Warp is easy to install, computationally inexpensive, and has an intuitive and streamlined user interface.
166 downloads biophysics
Although molecular self-organization and pattern formation are key features of life, only very few pattern-forming biochemical systems have been identified that can be reconstituted and studied in vitro under defined conditions. A systematic understanding of the underlying mechanisms is often hampered by multiple interactions, conformational flexibility and other complex features of the pattern forming proteins. Because of its compositional simplicity of only two proteins and a membrane, the MinDE system from Escherichia coli has already been invaluable for deciphering the mechanisms of spatiotemporal self-organization in cells. Here we explored the potential of reducing the complexity of this system even further, by identifying key functional motifs in the effector MinE that could be used to design pattern formation from scratch. In a combined approach of experiment and quantitative modeling, we show that starting from a minimal MinE-MinD interaction motif, pattern formation can be obtained by adding either dimerization or membrane-binding motifs.
162 downloads biophysics
Tracking the localization and mobility of individual proteins in live cells is key for understanding how they mediate their function. Such information can be obtained from single molecule imaging techniques such as Single Particle Tracking (SPT) and Single Molecule Localization Microscopy (SMLM). Genetic code expansion (GCE) combined with bioorthogonal chemistry offers an elegant approach for direct labeling of proteins with fluorescent dyes, holding great potential for improving protein labeling in single molecule applications. Here we calibrated conditions for performing SPT and live-SMLM of bioorthogonally labeled plasma membrane proteins in live mammalian cells. Using SPT, the diffusion rates of bioorthogonally labeled EGF receptor and the prototypical Shaker voltage-activated potassium channel (Kv) were successfully measured. Applying live-SMLM to bioorthogonally labeled Shaker Kv channels enabled visualizing the plasma membrane distribution of the channel over time with ~30 nm accuracy. Finally, by competitive labeling with two Fl-dyes, SPT and live-SMLM were performed in a single cell and both the density and dynamics of the EGF receptor were measured at single molecule resolution in sub-regions of the cell. We conclude that GCE and bioorthogonal chemistry is a highly suitable, flexible approach for protein labeling in quantitative single molecule applications that outperforms current protein live cell labeling approaches.
160 downloads biophysics
Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the C. crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multi-step assembly pathway. This pathway involves two domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2D crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using time-resolved electron cryo-microscopy (Cryo-EM) reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the two domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.
157 downloads biophysics
Anum A. Glasgow, Yao-Ming Huang, Daniel J. Mandell, Michael Thompson, Ryan Ritterson, Amanda L. Loshbaugh, Jenna Pellegrino, Cody Krivacic, Roland A. Pache, Kyle Barlow, Noah Ollikainen, Deborah Jeon, Mark J S Kelly, James S. Fraser, Tanja Kortemme
Sensing and responding to signals is a fundamental ability of living systems, but despite remarkable progress in computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here we describe a generalizable computational strategy for designing sensor/actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation via split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site matches the design model with atomic accuracy. Our computational design strategy opens broad avenues to link biological outputs to new signals.
156 downloads biophysics
The fast development of single particle cryo-EM has made it more feasible to obtain the 3D structure of well-behaved macromolecules with molecular weight higher than 300 kDa at ~3 Å resolution. It remains a challenge to obtain high resolution structure of molecules smaller than 100 kDa using single particle cryo-EM, mainly due to the low contrast of the molecules embedded in vitreous ice. In this work, we applied the Cs-corrector-VPP coupled cryo-EM to study 52 kDa streptavidin (SA) protein supported on a thin layer of graphene film and embedded in vitreous ice. We were able to solve both the apo-SA and biotin-bound SA at near-atomic resolution using single particle cryo-EM. We demonstrated that the method is capable to determine the structure of molecule as small as 39 kDa and potentially even smaller molecules. Furthermore, we found that using the graphene film to avoid the adsorption to the air-water interface is critical to maintain the protein's high-resolution structural information.
154 downloads biophysics
Morphogenesis of epithelial tissues requires tight spatiotemporal coordination of cell shape changes. In vivo, many tissue-scale shape changes are driven by pulsatile contractions of intercellular junctions, which are rectified to produce irreversible deformations. The functional role of this pulsatory ratchet and its mechanistic basis remain unknown. Here we combine theory and biophysical experiments to show that mechanosensitive tension remodelling of epithelial cell junctions promotes robust epithelial shape changes via ratcheting. Using optogenetic control of actomyosin contractility, we find that epithelial junctions show elastic behaviour under low contractile stress, returning to their original lengths after contraction, but undergo irreversible deformation under higher magnitudes of contractile stress. Existing vertex-based models for the epithelium are unable to capture these results, with cell junctions displaying purely elastic or fluid-like behaviours, depending on the choice of model parameters. To describe the experimental results, we propose a modified vertex model with two essential ingredients for junction mechanics: thresholded tension remodelling and continuous strain relaxation. First, a critical strain threshold for tension remodelling triggers irreversible junction length changes for sufficiently strong contractions, making the system robust to small fluctuations in contractile activity. Second, continuous strain relaxation allows for mechanical memory removal, enabling frequency-dependent modulation of cell shape changes via mechanical ratcheting. Taken together, the combination of mechanosensitive tension remodelling and junctional strain relaxation provides a robust mechanism for large-scale morphogenesis.
153 downloads biophysics
Substantial advances have been made in the computational design of protein interfaces over the last 20 years. However, the interfaces targeted by design have typically been stable and high affinity. Here, we report the development of a generic computational design method to stabilize the weak interactions at crystallographic interfaces. Initially, we analyzed structures reported in the Protein Data Bank (PDB) to determine whether crystals with more stable interfaces result in higher resolution structures. We found that, for twenty-two variants of a single protein crystallized by a single individual, Rosetta score correlates with resolution. We next developed and tested a computational design protocol, seeking to identify point mutations that would improve resolution on a highly stable variant of staphylococcal nuclease (SNase Δ+PHS). Only one of eleven initial designs crystallized, forcing us to re-evaluate our strategy and base our designs on an ensemble of protein backbones. Using this strategy, four of the five designed proteins crystallized. Collecting diffraction data for multiple crystals per design and solving crystal structures, we found that designed crystals improved resolution modestly and in unpredictable ways, including altering crystal space group. Post-hoc, in silico analysis showed that crystal space groups could have been predicted for four of six variants (including WT), but that resolution did not correlate with interface stability, as it did in the preliminary results. Our results show that single point mutations can have significant effects on crystal resolution and space group, and that it is possible to computationally identify such mutations, suggesting a potential design strategy to generate high-resolution protein crystals from poorly diffracting ones.
152 downloads biophysics
Thorsten Wagner, Felipe Merino, Markus Stabrin, Toshio Moriya, Claudia Antoni, Amir Apelbaum, Philine Hagel, Oleg Sitsel, Tobias Raisch, Daniel Prumbaum, Dennis Quentin, Daniel Roderer, Sebastian Tacke, Birte Siebolds, Evelyn Schubert, Tanvir R Shaikh, Pascal Lill, Christos Gatsogiannis, Stefan Raunser
Selecting particles from digital micrographs is an essential step in single particle electron cryomicroscopy (cryo-EM). Since manual selection of complete datasets typically comprising many thousands of particles is a tedious and time-consuming process, many automatic particle pickers have been developed in the past few decades. However, non-ideal datasets pose a challenge to particle picking. Here, we present a novel automated particle picking software called crYOLO, which is based on the deep learning object detection system 'You Only Look Once' (YOLO). After training the network with 500 - 2,500 particles per dataset, it automatically recognizes particles with high recall and precision reaching a speed of up to five micrographs per second. Importantly, we demonstrate a powerful general network trained on more than 40 datasets to select previously unseen datasets, thus paving the way for completely automated 'on-the-fly' cryo-EM data pre-processing during data acquisition. CrYOLO is available as a standalone program under http://sphire.mpg.de/ and will be part of the image processing workflow in SPHIRE.
152 downloads biophysics
Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogen Mycobacterium tuberculosis (Mtb). While this lipid promotes the entry of Mtb into macrophages, which occurs via phagocytosis, its molecular mechanism of action is unknown. Here, we combined biophysical, cell biology, and modelling approaches to reveal the molecular mechanism of DIM action on macrophage membranes leading to the first step of Mtb infection. MALDI-TOF mass spectrometry showed that DIM molecules are transferred from the Mtb envelope to macrophage membranes during infection. Multi-scale molecular modeling and 31P-NMR experiments revealed that DIM adopts a conical shape in membranes and aggregate in the stalks formed between two opposing lipid bilayers. Infection of macrophages pre-treated with lipids of various shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken together, these results reveal how the molecular shape of a mycobacterial lipid can modulate the biological function of macrophages.
151 downloads biophysics
Cell shapes and connectivities evolve over time as colony shapes change or embryos develop. Shapes of intercellular interfaces are closely coupled with the forces resulting from actomyosin interactions, membrane tension, or cell-cell adhesion. While it is possible to computationally infer cell-cell forces from a mechanical model of collective cell behavior, doing so for temporally evolving forces in a manner that is robust to digitization difficulties is challenging. Here, we introduce a method for Dynamic Local Intercellular Tension Estimation (DLITE) that infers such temporal force evolutions with less sensitivity to digitization ambiguities or errors. This method builds upon prior work on single time points (CellFIT). We validate our method using synthetic geometries. DLITE inferred cell colony tension evolutions correlate better with ground truth for these synthetic geometries than tension values inferred from methods that consider each time point in isolation. We introduce cell connectivity errors, angle estimate errors, connection mislocalization, and connection topological changes to synthetic data and show that DLITE has reduced sensitivity to these conditions. Finally, we apply DLITE to time series of human induced pluripotent stem (hIPS) cell colonies with endogenously expressed GFP-tagged ZO-1. We find major topological changes in cell connectivity, e.g. mitosis, can result in an increase in tension. This supports a correlation between the dynamics of cell-cell forces and colony rearrangement.
149 downloads biophysics
Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited its widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids are suitable for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and providing partial protection from the air-water interface. Additionally, the signal from images of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.
144 downloads biophysics
Imaging dense and diverse microbial communities has broad applications in basic microbiology and medicine, but remains a grand challenge due to the fact that many species adopt similar morphologies. While prior studies have relied on techniques involving spectral labeling, we have developed an expansion microscopy method (µExM) in which cells are physically expanded prior imaging and their expansion patterns depend on the structural and mechanical properties of their cell walls, which vary across species and conditions. We use this phenomenon as a quantitative and sensitive phenotypic imaging contrast orthogonal to spectral separation in order to resolve bacterial cells of different species or in distinct physiological states. Focusing on host-microbe interactions that are difficult to quantify through fluorescence alone, we demonstrate the ability of µExM to distinguish species within a dense community through in vivo imaging of a model gut microbiota, and to sensitively detect cell-envelope damage caused by antibiotics or previously unrecognized cell-to-cell phenotypic heterogeneity among pathogenic bacteria as they infect macrophages.
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