Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 55,041 bioRxiv papers from 254,018 authors.
Most downloaded bioRxiv papers, since beginning of last month
in category biophysics
2,198 results found. For more information, click each entry to expand.
697 downloads biophysics
Single-molecule localization microscopy (SMLM) promises to provide truly molecular scale images of biological specimens. However, mechanical instabilities in the instrument, readout errors and sample drift constitute significant challenges and severely limit both the useable data acquisition length and the localization accuracy of single molecule emitters. Here, we developed an actively stabilized total internal fluorescence (TIRF) microscope that performs 3D real-time drift corrections and achieves a stability of ≤1 nm. Self-alignment of the emission light path and corrections of readout errors of the camera automate channel alignment and ensure localization precisions of 1-4 nm in DNA origami structures and cells for different labels. We used Feedback SMLM to measure the separation distance of signaling receptors and phosphatases in T cells. Thus, an improved SMLM enables direct distance measurements between molecules in intact cells on the scale between 1-20 nm, potentially replacing Forster resonance energy transfer (FRET) to quantify molecular interactions. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.
664 downloads biophysics
HIV-1 Gag protein self-assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly the phosphatidylinositol (4,5) bisphosphate, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites was determined using super-resolution STED microscopy coupled with scanning Fluorescence Correlation Spectroscopy in living T cells. Analysis of HIV-1 infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data show that Gag is the main driving force to restrict PI(4,5)P2 and cholesterol mobility at the cell plasma membrane. This is first direct evidence showing that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol, as a membrane nano-platform for virus assembly.
532 downloads biophysics
The glucagon receptor family comprises Class B G protein-coupled receptors (GPCRs) that play a crucial role in regulating blood sugar levels. Receptors of this family represent important therapeutic targets for the treatment of diabetes and obesity. Despite intensive structural studies, we only have a poor understanding of the mechanism of peptide hormone-induced Class B receptor activation. This process involves the formation of a sharp kink in transmembrane helix 6 that moves out to allow formation of the nucleotide-free G protein complex. Here, we present the cryo-EM structure of the glucagon receptor (GCGR), a prototypical Class B GPCR, in complex with an engineered soluble glucagon derivative and the heterotrimeric G-protein, Gs. Comparison with the previously determined crystal structures of GCGR bound to a partial agonist reveals a structural framework to explain the molecular basis of ligand efficacy that is further supported by mutagenesis data.
447 downloads biophysics
Despite their great potential to facilitate rapid preparation of quite impure samples, affinity grids have not yet been widely employed in single particle cryo-EM. Here, we chemically functionalize graphene oxide coated grids and use a highly specific covalent affinity tag system. Importantly, our polyethylene glycol spacer keeps particles away from the air-water interface and graphene oxide surface, protecting them from denaturation or aggregation and permits high-resolution reconstructions of small particles.
430 downloads biophysics
Mucus clearance constitutes the primary defence of the respiratory system against viruses, bacteria and environmental insults. This transport across the entire airway emerges from the integrated activity of thousands of multiciliated cells, each containing hundreds of cilia, which together must coordinate their spatial arrangement, alignment and motility. The mechanisms of fluid transport have been studied extensively at the level of an individual cilium, collectively moving metachronal waves, and more generally the hydrodynamics of active matter. However, the connection between local cilia architecture and the topology of the flows they generate remains largely unexplored. Here, we image the mouse airway from the sub-cellular (nm) to the organ scales (mm), characterising quantitatively its ciliary arrangement and the generated flows. Locally we measure heterogeneity in both cilia organisation and flow structure, but across the trachea fluid transport is coherent. To examine this result, a hydrodynamic model was developed for a systematic exploration of different tissue architectures. Surprisingly, we find that disorder enhances particle clearance, whether it originates from fluctuations, heterogeneity in multiciliated cell arrangement or ciliary misalignment. This resembles elements of "stochastic resonance" in a self-assembled biological system. Taken together, our results shed light on how the microstructure of an active carpet determines its emergent dynamics. Furthermore, this work is also directly applicable to human airway pathologies, which are the third leading cause of deaths worldwide.
355 downloads biophysics
Inspired by the patterns of multicellularity in choanoflagellates, the closest living relatives of animals, we quantify the biophysical processes underlying the morphogenesis of rosette colonies in the choanoflagellate Salpingoeca rosetta. We find that rosettes reproducibly transition from an early stage of 2D growth to a later stage of 3D growth, despite the underlying stochasticity of the cell lineages. We postulate that the extracellular matrix (ECM) exerts a physical constraint on the packing of proliferating cells, thereby sculpting rosette morphogenesis. Our perturbative experiments coupled with biophysical simulations demonstrates the fundamental importance of a basally-secreted ECM for rosette morphogenesis. In addition, this yields a morphospace for the shapes of these multicellular colonies, consistent with observations of a range of choanoflagellates. Overall, our biophysical perspective on rosette development complements previous genetic perspectives and thus helps illuminate the interplay between cell biology and physics in regulating morphogenesis.
345 downloads biophysics
Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher-resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 Å compared to previous RELION versions.
326 downloads biophysics
RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. We used single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remained bound to DNA and sometimes exhibited one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarted transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this "secondary initiation" in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.
311 downloads biophysics
The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide controlled and efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving 4 standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.
280 downloads biophysics
Pedro P Vallejo Ramirez, Joseph Zammit, Oliver Vanderpoorten, Fergus Riche, Francois-Xavier Blé, Xiao-Hong Zhou, Bogdan F Spiridon, Christopher Valentine, Simeon P Spasov, Pelumi W Oluwasanya, Gemma Goodfellow, Marcus J. Fantham, Omid Siddiqui, Farah Alimagham, Miranda Robbins, Andrew Stretton, Dimitrios Simatos, Oliver Hadeler, Eric J. Rees, Florian Ströhl, Romain F Laine, Clemens F. Kaminski
The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of imaging large transparent specimens up to 13 mm tall and 8 mm deep with 50 μm resolution. OptiJ is based on off-the-shelf, easy-to-assemble optical components and an ImageJ plugin library for OPT data reconstruction. The software includes novel correction routines for uneven illumination and sample jitter in addition to CPU/GPU accelerated reconstruction for large datasets. We demonstrate the use of OptiJ to image and reconstruct cleared lung lobes from adult mice. We provide a detailed set of instructions to set up and use the OptiJ framework. Our hardware and software design are modular and easy to implement, allowing for further open microscopy developments for imaging large organ samples.
252 downloads biophysics
Although microscopes and image analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here we present a simple cryo-EM specimen preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. The device is controlled by a Raspberry Pi single board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM, rather than instrument design. The simple open-source design permits straightforward customization of the instrument for specialized experiments.
224 downloads biophysics
CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and Gaussian accelerated Molecular Dynamics (GaMD) simulations - flanked by mixed machine learning and structure-based prediction of NMR chemical shifts - are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond timescale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in the full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome editing capability
223 downloads biophysics
Multi-emitter localization has great potential for maximizing the imaging speed of super-resolution localization microscopy. However, the slow image analysis speed of reported multi-emitter localization algorithms limits their usage in mostly off-line image processing with small image size. Here we adopt the well-known divide and conquer strategy in computer science and present a fitting-based method called QC-STORM for fast multi-emitter localization. Using simulated and experimental data, we verify that QC-STORM is capable of providing real-time full image processing on raw images with 100 um x 100 um field of view and 10 ms exposure time, with comparable spatial resolution as the popular fitting-based ThunderSTORM and the up-to-date non-iterative WindSTORM. This study pushes the development and practical use of super-resolution localization microscopy in high-throughput or high-content imaging of cell-to-cell differences or discovering rare events in a large cell population.
213 downloads biophysics
Every organ surface and body cavity is lined by a confluent collective of epithelial cells. In homeostatic circumstances the epithelial collective remains effectively solid-like and sedentary. But during morphogenesis, remodeling or repair, as well as during malignant invasion or metastasis, the epithelial collective becomes fluid-like and migratory. This conversion from sedentary to migratory behavior has traditionally been understood as a manifestation of the epithelial-to-mesenchymal transition (EMT) or the partial EMT (pEMT). However, in certain contexts this conversion has been attributed to the recently discovered unjamming transition (UJT), in which epithelial cells move collectively and cooperatively. UJT and pEMT share certain aspects of collective cellular migration, but the extent to which these processes are distinct, overlapping or perhaps even identical has remained undefined. Using the confluent layer of well-differentiated primary human bronchial epithelial (HBE) cells, here we triggered UJT by exposing the sedentary layer to mechanical compression that mimics the mechanical environment of asthmatic bronchoconstricion. Cells thereafter migrated cooperatively, aligned into packs locally, and elongated systematically. Nevertheless, cell-cell junctions, apico-basal polarity, and barrier function remained intact in response, and mesenchymal markers remained unapparent. As such, pEMT was not evident. When we triggered pEMT and associated cellular migration by exposing the sedentary layer to TGF-β1, metrics of UJT versus pEMT diverged. To account for these striking physical observations a new mathematical model attributes the effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Together, these findings establish that UJT is sufficient to account for vigorous epithelial layer migration even in the absence of pEMT. Distinct gateways to cellular migration therefore become apparent - UJT as it might apply to migration of epithelial sheets, and EMT/pEMT as it might apply to migration of mesenchymal cells on a solitary or collective basis, activated during development, remodeling, repair or tumor invasion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, living tissue is therefore seen to comprise an active engineering material whose modules for plasticity, self-repair and regeneration, are far richer than had been previously appreciated.
212 downloads biophysics
Fast neurotransmission is mediated by pentameric ligand-gated ion channels. Glycine receptors are chloride-selective members of this receptor family that mediate inhibitory synaptic transmission and are implicated in neurological disorders including autism and hyperekplexia. They have been structurally characterized by both X-ray crystallography and cryo electron microscopy studies, with the latter giving rise to what was proposed as a possible open state. However, recent work has questioned the physiological relevance of this open state structure, since it rapidly collapses in molecular dynamics simulations. Here, we show that the collapse can be avoided by a careful equilibration protocol that reconciles the more problematic regions of the original electron-density map and gives a stable open state that shows frequent selective chloride permeation. The protocol developed in this work provides a means to refine open-like structures of the whole pentameric ligand-gated ion channel superfamily and reconciles the previous issues with the cryo-EM structure.
202 downloads biophysics
All cellular structures are assembled from molecular building blocks, and molecular building blocks incur energetic costs to the cell. In an energy-limited environment, the energetic cost of a cellular structure imposes a fitness cost and impacts a cell's evolutionary trajectory. While the importance of energetic considerations was realized for decades, the distinction between direct energetic costs expended by the cell and potential energy that the cell diverts into cellular biomass components, which we define as the opportunity cost, was not explicitly made, leading to large differences in values for energetic costs of molecular building blocks used in the literature. We describe a framework that defines and separates various components relevant for estimating the energetic costs of molecular building blocks and the resulting cellular structures. This distinction among energetic costs is an essential step towards discussing the conversion of an energetic cost to a corresponding fitness cost.
194 downloads biophysics
We describe a method for quantifying the contractile forces that tumor spheroids collectively exert on highly nonlinear three-dimensional collagen networks. While three-dimensional traction force microscopy for single cells in a nonlinear matrix is computationally complex due to the variable cell shape, here we exploit the spherical symmetry of tumor spheroids to derive a scale-invariant relationship between spheroid contractility and the surrounding matrix deformations. This relationship allows us to directly translate the magnitude of matrix deformations to the total contractility of arbitrarily sized spheroids. We show that collective forces of tumor spheroids reflect the contractility of individual cells for up to 1h after seeding, while collective forces on longer time-scales are guided by mechanical feedback from the extracellular matrix.
187 downloads biophysics
Magnetoreception, the perception of the geomagnetic field, is a sensory modality well-established across all major groups of vertebrates and some invertebrates, but its presence in humans has been tested rarely, yielding inconclusive results. We report here a strong, specific human brain response to ecologically-relevant rotations of Earth-strength magnetic fields. Following geomagnetic stimulation, a drop in amplitude of EEG alpha oscillations (8-13 Hz) occurred in a repeatable manner. Termed alpha event-related desynchronization (alpha-ERD), such a response is associated with sensory and cognitive processing of external stimuli. Biophysical tests showed that the neural response was sensitive to the dynamic components and axial alignment of the field but also to the static components and polarity of the field. This pattern of results implicates ferromagnetism as the biophysical basis for the sensory transduction and provides a basis to start the behavioral exploration of human magnetoreception.
185 downloads biophysics
Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are cation channels that mobilize Ca2+ from intracellular stores in response to a wide range of cellular stimuli. The paradigm of InsP3R activation is the coupled interplay between binding of InsP3 and Ca2+ that switches the ion conduction pathway between closed and open states to enable the passage of Ca2+ through the channel. However, the molecular mechanism of how the receptor senses and decodes ligand-binding signals into gating motion remains unknown. Here we present the electron cryo-microscopy structure of InsP3R1 from rat cerebellum determined to 4.1 Å resolution in the presence of activating concentrations of Ca2+ and adenophostin A (AdA), a structural mimetic of InsP3 and the most potent known agonist of the channel. Comparison with the 3.9 Å-resolution structure of InsP3R1 in the Apo-state, also reported herein, reveals the binding arrangement of AdA in the tetrameric channel assembly and striking ligand-induced conformational rearrangements within cytoplasmic domains coupled to the dilation of a hydrophobic constriction at the gate. Together, our results provide critical insights into the mechanistic principles by which ligand-binding allosterically gates InsP3R channel.
183 downloads biophysics
Severe injury to the mammalian spinal cord results in permanent loss of function due to the formation of a glial-fibrotic scar. Both the chemical composition and the mechanical properties of the scar tissue have been implicated to inhibit neuronal regrowth and functional recovery. By contrast, adult zebrafish are able to repair spinal cord tissue and restore motor function after complete spinal cord transection owing to a complex cellular response that includes neurogenesis and axon regrowth. The mechanical mechanisms contributing to successful spinal cord repair in adult zebrafish are, however, currently unknown. Here, we employ AFM-enabled nano-indentation to determine the spatial distributions of apparent elastic moduli of living spinal cord tissue sections obtained from uninjured zebrafish and at distinct time points after complete spinal cord transection. In uninjured specimens, spinal gray matter regions were stiffer than white matter regions. During regeneration after transection, the spinal cord tissues displayed a significant increase of the respective apparent elastic moduli that transiently obliterated the mechanical difference between the two types of matter, before returning to baseline values after completion of repair. Tissue stiffness correlated variably with cell number density, oligodendrocyte interconnectivity, axonal orientation, and vascularization. The presented work constitutes the first quantitative mapping of the spatio-temporal changes of spinal cord tissue stiffness in regenerating adult zebrafish and provides the tissue mechanical basis for future studies into the role of mechanosensing in spinal cord repair.
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