Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 62,303 bioRxiv papers from 276,577 authors.
Most downloaded bioRxiv papers, since beginning of last month
in category biophysics
2,071 results found. For more information, click each entry to expand.
278 downloads biophysics
The development of novel analgesics with improved safety profiles to combat the opioid epidemic represents a central question to G protein coupled receptor structural biology and pharmacology: What chemical features dictate G protein or β-arrestin signaling? Here we use adaptively biased molecular dynamics simulations to determine how fentanyl, a potent β-arrestin biased agonist, activates the μ-opioid receptor (μOR). The resulting fentanyl-bound pose provides rational insight into a wealth of historical structure-activity-relationship on its chemical scaffold. We found that fentanyl and the synthetic opioid peptide DAMGO require M153 to induce β-arrestin coupling, while M153 was dispensable for G protein coupling. We propose and validate a mechanism where the n-aniline ring of fentanyl mediates μOR β-arrestin through a novel M153 “microswitch” by synthesizing fentanyl-based derivatives that exhibit complete, clinically desirable, G protein biased coupling. Together, these results provide molecular insight into fentanyl mediated β-arrestin biased signaling and a rational framework for further optimization of fentanyl-based analgesics with improved safety profiles.
276 downloads biophysics
Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental and therapeutic application has been difficult for lack of agents amenable to tissue targeting. We have developed a novel agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1. This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix", as revealed by cryo-EM structure determination. Although this conformation likely constitutes part of the transport cycle, nanobody-trapping disrupts the cycle and prevents substrate movement through the Patched1 sterol conduit. Our conformation-selective nanobody approach provides a new route to the development of transporter-related pharmacologic agents and may be generally applicable to the study of other transporters.
267 downloads biophysics
Amanda Buyan, Charles Cox, James Rae, Jonathan Barnoud, Jinyuan Li, Jasmina Cvetovska, Michele Bastiani, Hannah SM Chan, Mark P Hodson, Boris Martinac, Robert G. Parton, Siewert-Jan Marrink, Ben Corry
Touch, hearing, and blood pressure control require mechanically-gated ion channels that convert mechanical stimuli into electrical currents. Piezo1 and Piezo2 were recently identified as essential eukaryotic mechanically-gated ion channels, yet how they respond to physical forces remains poorly understood. Here we use a multi-disciplinary approach to interrogate the interaction of Piezo1 with its lipid environment. We show that individual Piezo1 channels induce significant local curvature in the membrane that is magnified in a cooperative manner to generate larger curved Piezo1 pits. Curvature decreases under lateral membrane tension, consistent with a hypothesis that force detection can involve sensing changes to local curvature. The protein alters its local membrane composition, enriching specific lipids and forming essential binding sites for phosphoinositides and cholesterol that are functionally relevant and often related to Piezo1-mediated pathologies. Finally, we show that Piezo1 alters the expression of lipid-regulating proteins and modifies the cellular lipidome. In short, we find that lipids influence Piezo1 activity and Piezo1 influences the local morphology and composition of the bilayer as well as the cellular lipidome.
259 downloads biophysics
In single-molecule localization based super-resolution microscopy (SMLM), a fluorophore stochastically switches between fluorescent- and dark-states, leading to intermittent emission of fluorescence. Intermittent emissions create multiple localizations belonging to the same molecule, a phenomenon known as blinking. Blinking distorts SMLM images and confound quantitative interpretations by forming artificial nanoclusters, which are often interpreted as true biological assemblies. Multiple methods have been developed to eliminate these artifacts, but they either require additional experiments, arbitrary thresholds, or specific photo-kinetic models. Here we present a method, termed Distance Distribution Correction (DDC), to eliminate fluorophore blinking in superresolution imaging without any additional calibrations. The approach relies on the finding that the true pairwise distance distribution of different fluorophores in an SMLM image can be naturally obtained from the imaging sequence by using the distances between localizations separated by a time much longer than the average fluorescence survival time. We show that using the true pairwise distribution we can define and then maximize the likelihood of obtaining a particular set of localizations without blinking and generate an accurate reconstruction of the true underlying cellular structure. Using both simulated and experimental data, we show that DDC surpasses all previous existing blinking correction methodologies, resulting in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters. The simplicity and robustness of DDC will enable its wide application in SMLM imaging, providing the most accurate reconstruction and quantification of SMLM images to date.
256 downloads biophysics
Our senses of hearing and balance rely on the extraordinarily sensitive molecular machinery of the inner ear to convert deflections as small as the width of a single carbon atom1,2 into electrical signals that the brain can process3. In humans and other vertebrates, transduction is mediated by hair cells4, where tension on tip links conveys force to mechanosensitive ion channels5. Each tip link comprises two helical filaments of atypical cadherins bound at their N-termini through two unique adhesion bonds6-8. Tip links must be strong enough to maintain a connection to the mechanotransduction channel under the dynamic forces exerted by sound or head movement - yet might also act as mechanical circuit breakers, releasing under extreme conditions to preserve the delicate structures within the hair cell. Previous studies have argued that this connection is exceptionally static, disrupted only by harsh chemical conditions or loud sound9-12. However, no direct mechanical measurements of the full tip-link connection have been performed. Here we describe the dynamics of the tip-link connection at single-molecule resolution and show how avidity conferred by its double stranded architecture enhances mechanical strength and lifetime, yet still enables it to act as a dynamic mechanical circuit breaker. We also show how the dynamic strength of the connection is facilitated by strong cis-dimerization and tuned by extracellular Ca2+, and we describe the unexpected etiology of a hereditary human deafness mutation. Remarkably, the connection is several thousand times more dynamic than previously thought, challenging current assumptions about tip-link stability and turnover rate, and providing insight into how the mechanotransduction apparatus conveys mechanical information. Our results reveal fundamental mechanisms that underlie mechanoelectric transduction in the inner ear, and provide a foundation for studying multi-component linkages in other biological systems.
254 downloads biophysics
Global changes of cell shape under mechanical or osmotic external stresses are mostly controlled by the mechanics of the cortical actin cytoskeleton underlying the cell membrane. Some aspects of this process can be recapitulated in vitro on reconstituted actin-and-membrane systems. In this paper, we investigate how the mechanical properties of a branched actin network shell, polymerized at the surface of a liposome, control membrane shape when the volume is reduced. We observe a variety of membrane shapes depending on the actin thickness. Thin shells undergo buckling, characterized by a cup-shape deformation of the membrane that coincides with the one of the actin network. Thick shells produce membrane wrinkles, but do not deform their outer layer. For intermediate micrometer-thick shells, wrinkling of the membrane is observed, and the actin layer is slightly deformed. Confronting our experimental results with a theoretical description, we determine the transition between buckling and wrinkling depending on the thickness of the actin shell and the size of the liposome. We thus unveil the generic mechanism by which biomembranes are able to accommodate their shape against mechanical compression, through thickness adaptation of their cortical cytoskeleton.
253 downloads biophysics
Developing biological structures are highly complex systems, within which a robust and efficient information transfer must happen to coordinate several dynamical processes. Through a reformulation of cytokinesis in terms of its energetic cost, we propose that oriented cell divisions are one mechanism by which cells can read and react to mechanical forces propagating in a tissue even in the absence of cellular strain. This view can at once account for the standard geometrical rules for cell division, as much as the known systematic deviations from them. Our results suggest the existence of a shape-dynamics to cell-division feedback loop, consisting in the competition between local and long-range mechanical signals. The consequences of this competition are explored in simulated tissues and confirmed in vivo during the epidermal morphogenesis of ascidian embryos.
250 downloads biophysics
Probing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED)-FCS. Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of more than 1 MHz) using novel detection equipment, namely hybrid detectors and realtime gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Other advantages include greater flexibility of experiments with biological samples with very highly variable intensity (e.g. due to a wide range of expression levels of fluorescent proteins), much shorter acquisition time, and improved data quality. This approach also pronouncedly increased the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics.
249 downloads biophysics
Ahmad Abdelzaher Zaki Khalifa, Muneyoshi Ichikawa, Daniel Dai, Shintaroh Kubo, Corbin Black, Katya Peri, Thomas S McAlear, Simon Veyron, Shun Kai Yang, Javier Vargas, Susanne Bechstedt, Jean-Francois Trempe, Khanh Huy Bui
Microtubules are cytoskeletal structures involved in structural support, microtubule-based transport and the organization of organelles in the cells. The building blocks of the microtubule, the α- and β-tubulin heterodimers, polymerize into protofilaments, that associate laterally to form the hollow microtubule. There exists a specific type of microtubule structures in the cilia, termed doublet microtubules, where high stability is required for ciliary beating and function. The doublet microtubule, consisting of a complete A-tubule and a partial B-tubule maintains its stability through unique interactions at its outer and inner junctions, where the A- and B-tubules meet. Using cryo-electron microscopy, we present the answer to the long-standing question regarding the identities, localizations and structures of the Chlamydomonas doublet microtubule inner junction proteins. Using a combination of sequence bioinformatics and mass spectrometry, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction associated protein FAP126. We show that inner junction proteins PACRG and FAP20, together with FAP52, previously unidentified FAP276, FAP106 and FAP126, form an interaction hub at the inner junction, which involves tubulin sites for post-translational modifications. We further compare the Chlamydomonas and Tetrahymena doublet microtubule structures to understand the common and species-specific features of the inner junction.
248 downloads biophysics
A continuous sheet of epithelial cells surrounding a hollow opening, or lumen, defines the basic topology of numerous organs. De novo lumen formation is a central feature of embryonic development whose dysregulation leads to congenital and acquired diseases of the kidney and other organs. While prior work has described the hydrostatic pressure-driven expansion of lumens when they are large, the physical mechanisms that promote the formation and maintenance of small, nascent lumens are less explored. In particular, models that rely solely on pressure-driven expansion face a potential challenge in that the Laplace pressure, which resists lumen expansion, is predicted to scale inversely with lumen radius. We investigated the cellular and physical mechanisms responsible for stabilizing the initial stages of lumen growth using a 3D culture system in which epithelial cells spontaneously form hollow lumens. Our experiments revealed that neither the actomyosin nor microtubule cytoskeletons are required to stabilize lumen geometry, and that a positive intraluminal pressure is not necessary for lumen stability. Instead, our observations are in agreement with a quantitative model in which cells maintain lumen shape due to topological and geometrical factors tied to the establishment of apico-basolateral polarity. We suggest that this model may provide a general physical mechanism for the formation of luminal openings in a variety of physiological contexts.
232 downloads biophysics
Cell migration is involved in key phenomena in biology, ranging from development to cancer. Fibroblasts move between organs in 3D polymeric networks. So far, motile cells were mainly tracked in vitro on Petri dishes or on coverslips, i.e. 2D flat surfaces, which made the extrapolation to 3D physiological environments difficult. We therefore prepared 3D Cell Derived Matrix (CDM) with specific characteristics with the goal of extracting the main readouts required to measure and characterise cell motion: cell specific matrix deformation through the tracking of fluorescent fibronectin within CDM, focal contacts as the cell anchor and acto-myosin cytoskeleton which applies cellular forces. We report our method for generating this assay of physiological-like gel with relevant readouts together with its potential impact in explaining cell motility in vivo.
226 downloads biophysics
Tissue elongation is a central morphogenetic event occurring in all organisms in development. During this process, symmetry of cells and tissues is broken by different mechanisms, such as neighbor exchange, cell elongation and oriented cell division. While the phenomenon is known to involve remodeling of adherens junctions and acto-myosin at the molecular level, mesoscopic mechanisms leading to distinct morphogenesis processes are poorly understood. This is partly because inputs from morphogen gradients or from neighboring tissues can affect tissue autonomous self-organization in vivo. It is therefore difficult to disentangle cell intrinsic from externally mediated behaviors. Here we use in vitro experiments and numerical simulations to characterize the spontaneous behavior of a growing cell colony in vitro. We show that in vitro tissue elongation arises from anisotropy in the average cell elongation. This anisotropy sets the direction along which boundary cells migrate radially resulting in a non-isotropic elongation that arises primarily through cell elongation. For colonies submitted to a time periodic stretch, the axis of global symmetry breaking can be imposed by external force, and tissue elongation arises through oriented neighbor exchange. Emergence of radially migrating cells and the interplay between cell elongation and cell rearrangements are confirmed by numerical simulations based on a vertex model. Our results suggest that spontaneous shape deformation is related to the mean orientation of the nematic cell elongation field in the absence of any external input. This provides a framework to explain autonomous tissue elongation and how contributions from different mesoscopic mechanisms can be modulated by external forces.
217 downloads biophysics
Bacteria have evolved adaptive immune systems encoded by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated (Cas) genes to maintain genomic integrity in the face of relentless assault from pathogens and mobile genetic elements. Type I CRISPR-Cas systems canonically target foreign DNA for degradation via the joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3, but nuclease-deficient Type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons. How CRISPR -and transposon-associated machineries collaborate during DNA targeting and insertion has remained elusive. Here we determined structures of a novel TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using single particle electron cryo-microscopy (cryo-EM), revealing the mechanistic basis of this functional coupling. The quality of the cryo-EM maps allowed for de novo modeling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the crRNA. The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer adjacent motif (PAM) recognition and R-loop formation. The present work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposon-associated proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome engineering applications.
215 downloads biophysics
Single-molecule localization microscopy super-resolution methods such as DNA-PAINT and (d)STORM can generate multiple observed localizations over the time course of data acquisition from each dye or binding site that are not a priori assigned to those specific dyes or binding sites. We describe a Bayesian method of grouping and combining localizations from multiple blinking/binding events that can improve localization precision to better than one nanometer. The known statistical distribution of the number of binding/blinking events per dye/docking strand along with the precision of each localization event are used to estimate the true number and location of emitters in closely-spaced clusters.
213 downloads biophysics
Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC=0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device. Data were collected using a compact, two-lens illumination system with a constant power objective lens, without the use of energy filters or aberration correctors. Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (x-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving resolution close to the Rayleigh limit. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging, widely thought of as a “screening cryo-microscope”, can be used to determine structures approaching atomic resolution. Highlights
208 downloads biophysics
Intrinsically disordered proteins or regions (IDRs) differ from their well-folded counterparts by lacking a stable tertiary state. Instead, IDRs exist in an ensemble of conformations and often possess localized, loosely held residual structure that can be a key determinant of their activity. With no extensive network of non-covalent bonds and a high propensity for exposed surface areas, the various features of an IDR's ensemble - including local residual structure and global conformational biases - are an emergent property of both the amino acid sequence and the solution environment. Here, we attempt to understand how shifting solution conditions can alter an IDR's ensemble. We present an efficient computational method to alter solution-protein interactions we term Solution Space (SolSpace) Scanning. SolSpace scanning uses all-atom Monte-Carlo simulations to construct ensembles under a wide range of distinct solution conditions. By tuning the interactions of specific protein moieties with the solution in a systematic manner we can both enhance and reduce local residual structure. This approach allows the 'design' of distinct residual structures in IDRs, offering an alternative approach to mutational studies for exploring sequence-to-ensemble relationships. Our results raise the possibility of solution-based regulation of protein functions both outside and within the dynamic solution environment of cells.
204 downloads biophysics
Mitochondria contain the genetic information and expression machinery to produce proteins essential for cellular respiration. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins, and mitoribosome assembly factors are known to form punctate subcompartments referred to as mitochondrial RNA granules (MRGs) 1-3. Despite their proposed role in regulating gene expression, little is known about the structural and dynamic properties of MRGs. We investigated the organization of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy techniques, obtaining ultrastructural details of their internal architecture. We find that MRGs are organized into nanoscale RNA cores surrounded by a protein shell. Using live-cell super-resolution structured illumination microscopy and photobleaching perturbations, we reveal that MRGs undergo fusion and rapidly exchange components, consistent with liquid-liquid phase separation (LLPS). Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with membrane remodeling. Inhibition of mitochondrial fission leads to an aberrant distribution of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our results reveal a role for LLPS in concentrating RNA and its processing proteins into MRGs, which are positioned along mitochondria by membrane dynamics.
199 downloads biophysics
Partition coefficients describe the equilibrium partitioning of a neutral solute between two immiscible phases. Octanol-water partition coefficients (Kow), or their logarithms (log P), are frequently used as a measure of lipophilicity in drug discovery. The partition coefficient is a physicochemical property that captures the thermodynamics of relative solvation between aqueous and nonpolar phases, and therefore provides an excellent test for physics-based computational models that predict properties of pharmaceutical relevance such as protein-ligand binding affinities or hydration/solvation free energies. The SAMPL6 Part II Octanol-Water Partition Coefficient Prediction Challenge used a subset of kinase inhibitor fragment-like compounds from the SAMPL6 pKa Prediction Challenge in a blind experimental benchmark. Following experimental data collection, the partition coefficient dataset was kept blinded until all predictions were collected from participating computational chemistry groups. A total of 91 submissions were received from 27 participating research groups. This paper presents the octanol-water log P dataset for this SAMPL6 Part II Partition Coefficient Challenge, which consisted of 11 compounds (six 4-aminoquinazolines, two benzimidazole, one pyrazolo[3,4-d]pyrimidine, one pyridine, one 2-oxoquinoline substructure containing compounds) with log P values in the range of 1.95-4.09. We describe the potentiometric log P measurement protocol used to collect this dataset using a Sirius T3, discuss the limitations of this experimental approach, and share suggestions for future log P data collection efforts for the evaluation of computational methods.
199 downloads biophysics
Solid-state nanopores are a single-molecule technique that can provide access to biomolecular information that is otherwise masked by ensemble averaging. A promising application uses pores and barcoding chemistries to map molecular motifs along single DNA molecules. Despite recent research breakthroughs, however, it remains challenging to overcome molecular noise to fully exploit single molecule data. Here we present an active control technique termed "flossing" that uses a dual nanopore device to trap a protein-tagged DNA molecule and perform up to 100's of back and-forth electrical scans of the molecule in a few seconds. The protein motifs bound to 48 kb lambda DNA are used as detectable features for active triggering of the bidirectional control. Molecular noise is suppressed by averaging the multi-scan data to produce averaged inter-tag distance estimates that are comparable to their known values. Since nanopore feature-mapping applications require DNA linearization when passing through the pore, a key advantage of flossing is that trans-pore linearization is increased to >98% by the second scan, compared to 35% for single nanopore passage of the same set of molecules. In concert with barcoding methods, the dual-pore flossing technique could enable genome mapping and structural variation applications, or mapping loci of epigenetic relevance.
195 downloads biophysics
Sonia Ciudad, Eduard Puig, Thomas Botzanowski, Moeen Meigooni, Andres S. Arango, Jimmy Do, Maxim Mayzel, Mariam Bayoumi, Stéphane Chaignepain, Giovanni Maglia, Sarah Cianferani, Vladislav Orekhov, Emad Tajkhorshid, Benjamin Bardiaux, Natàlia Carulla
The formation of amyloid-beta (Aβ) oligomer pores in the membrane of neurons has been proposed as the means to explain neurotoxicity in Alzheimer's disease (AD). It is therefore critical to characterize Aβ oligomer samples in membrane-mimicking environments. Here we present the first three-dimensional structure of an Aβ oligomer formed in dodecyl phosphocholine (DPC) micelles, namely an Aβ(1-42) tetramer. It comprises a β-sheet core made of six β-strands, connected by only two β-turns. The two faces of the β-sheet core are hydrophobic and surrounded by the membrane-mimicking environment. In contrast, the edges of the core are hydrophilic and are solvent-exposed. By increasing the concentration of Aβ(1-42), we prepared a sample enriched in Aβ(1-42) octamers, formed by two Aβ(1-42) tetramers facing each other forming a β-sandwich structure. Notably, samples enriched in Aβ(1-42) tetramers and octamers are both active in lipid bilayers and exhibit the same types of pore-like behaviour, but they show different occurrence rates. Remarkably, molecular dynamics simulations showed a new mechanism of membrane disruption in which water and ion permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core β-sheets edges of the Aβ(1-42) tetramers and octamers.
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