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bioRxiv2017

NanoJ-SQUIRREL: quantitative mapping and minimisation of super-resolution optical imaging artefacts

Most super-resolution microscopy methods depend on steps that contribute to the formation of image artefacts. Here we present NanoJ-SQUIRREL, an ImageJ-based analytical approach providing a quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same focal volume, this approach generates a quantitative map of super-resolution defects, as well as methods for their correction. To illustrate its broad applicability to super-resolution approaches we apply our method to Localization Microscopy, STED and SIM images of a variety of in-cell structures including microtubules, poxviruses, neuronal actin rings and clathrin coated pits. We particularly focus on single-molecule localisation microscopy, where super-resolution reconstructions often feature imperfections not present in the original data. By showing the quantitative evolution of data quality over these varied sample preparation, acquisition and super-resolution methods we display the potential of NanoJ-SQUIRREL to guide optimization of superresolution imaging parameters.

biophysics↗

bioRxiv2017

Activation and desensitization mechanism of AMPA receptor-TARP complex by cryo-EM

Summary Summary Structure determination Agonists expand LBD gating... Activation by full and... Architecture of the ion... Ion-permeation profile of the... Asymmetric water/ion accessible... TARP modulation of receptor... TARP restricts LBD rearrangement... TARP modulation of AMPA... Materials and methods AUTHOR CONTRIBUTIONS AUTHOR INFORMATION References AMPA receptors mediate the majority of fast excitatory neurotransmission in the mammalian brain and transduce the binding of presynaptically released glutamate to the opening of a transmembrane cation channel. Within the postsynaptic density, however, AMPA receptors coassemble with transmembran ...

biophysics↗

bioRxiv2017

Transferable tICA-Metadynamics: Efficient sampling of protein mutants by transferring information from the wild type’s Markov state model

We recently showed that the time-structure based independent component analysis method from Markov state model literature provided a set of variationally optimal slow collective variables for Metadynamics (tICA-Metadynamics). In this paper, we extend the methodology towards efficient sampling of related mutants by borrowing ideas from transfer learning methods in machine learning. Our method explicitly assumes that a similar set of slow modes and metastable states are found in both the wild type (base line) and its mutants. Under this assumption, we describe a few simple techniques using sequence mapping for transferring the slow modes and structural information contained in the wild type simulation to a mutant model for performing enhanced sampling. The resulting simulations can then be reweighted onto the full-phase space using Multi-state Bennett Acceptance Ratio, allowing for thermodynamic comparison against the wild type. We first benchmark our methodology by re-capturing alanine dipeptide dynamics across a range of different atomistic force fields, including the polarizable Amoeba force field, after learning a set of slow modes using Amber ff99sb-ILDN. We next extend the method by including structural data from the wild type simulation and apply the technique to recapturing the affects of the GTT mutation on the FIP35 WW domain.

biophysics↗

bioRxiv2017

A Fluorogenic Array Tag for Temporally Unlimited Single Molecule Tracking

Cellular processes take place over many timescales, prompting the development of precision measurement technologies that cover milliseconds to hours. Here we describe ArrayG, a bipartite fluorogenic system composed of a GFP-nanobody array and monomeric wtGFP binders. The free binders are initially dim but brighten 15 fold upon binding the array, suppressing background fluorescence. By balancing rates of intracellular binder production, photo-bleaching, and stochastic binder exchange on the array, we achieved temporally unlimited tracking of single molecules. Fast (20-180Hz) tracking of ArrayG tagged kinesins and integrins, for thousands of frames, revealed repeated state-switching and molecular heterogeneity. Slow (0.5 Hz) tracking of single histones for as long as 1 hour showed fractal dynamics of chromatin. We also report ArrayD, a DHFR-nanobody-array tag for dual color imaging. The arrays are aggregation resistant and combine high brightness, background suppression, fluorescence replenishment, and extended choice of fluorophores, opening new avenues for seeing and tracking single molecules in living cells.

biophysics↗

bioRxiv2017

Single-cell absolute contact probability detection reveals that chromosomes are organized by modulated stochasticity

At the kilo- to mega-base pair scales, eukaryotic genomes are partitioned into self-interacting modules or topologically associated domains (TADs) that associate to form nuclear compartments. Here, we combined high-content super-resolution microscopies with state-of-the-art DNA labeling methods to reveal the variability in the multiscale organization of the Drosophila genome. We found that association frequencies within TADs and between TAD borders are below ~10%, independently of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, we were able to visualize nanometer-sized epigenetic domains at the single-cell level. In addition, absolute contact frequencies within and between TADs were to a large extent defined by genomic distance, higher-order chromosome architecture, and epigenetic identity. We propose that TADs and compartments are organized by multiple, small frequency, yet specific interactions that are regulated by epigenetics and transcriptional state.

biophysics↗

bioRxiv2017

Mechanics of Hydra Detachment from Subrates: The Role of Substrate Rigidity and Starvation

Hydra is a fresh water hydrozoan living as a solitary polyp with a sedentary feeder lifestyle attached to a substrate. In times of food shortage they are reported to detach from their substrate and move either by drifting or somer-saulting. The attachment to the substrate is usually by the basal-body which secretes a mucosal adhesive. The mechanical strength of the adhesion of Hydra has not been quantified so far. Here, we measure the force required to detach Hydra vulgaris and Hydra magnipapillata from a surface and the role of physical and physiological factors. In order to do this, we have developed a flow chamber with a calibrated jet of water. We find H. vulgaris adhering to a hard substrate - a glass cover slip- requires more force to detach it as compared to a soft substrate- polyacrylamide gel. While H. vulgaris after one week of starvation detaches with very similar values of stress, H. magnipapillata detaches more readily when starved. These results suggest that the strength of adhesion is strongly affected by the stiffness of the substrate, while nutritional status dependence of detachment force appears to be species dependent. Given that Hydra detachment is required during locomotion, our measurements on the one hand suggest the magnitude of forces the animal must exert to detach itself. Additionally, our results suggest active detachment of the base might be required for Hydra to achieve movement, and only a small contribution coming from weakening adhesion.

biophysics↗

bioRxiv2017

Pavement cells and the topology puzzle

DArcy Thompson emphasised the importance of surface tension as a potential driving force in establishing cell shape and topology within tissues. Leaf epidermal pavement cells grow into jigsaw-piece shapes, highly deviating from such classical forms. We investigate the topology of developing Arabidopsis leaves composed solely of pavement cells. Image analysis of around 50,000 cells reveals a clear and unique topological signature, deviating from previously studied epidermal tissues. This topological distribution is however established early during leaf development, already before the typical pavement cell shapes emerge, with topological homestasis maintained throughout growth and unaltered between division and maturation zones. Simulating graph models, we identify a heuristic cellular division rule that reproduces the observed topology. Our parsimonious model predicts how and when cells effectively place their division plane with respect to their neighbours. We verify the predicted dynamics through in vivo tracking of 800 mitotic events, and conclude that the distinct topology is not a direct consequence of the jigsaw-like shape of the cells, but rather owes itself to a strongly life-history-driven process, with limited impact from cell surface mechanics.\n\nSummary statement\n\nDevelopment of the Arabidopsis leaf epidermis topology is driven by deceptively simple rules of cell division, independent of surface tension, cell size and, often complex, cell shape.

biophysics↗

bioRxiv2017

A computational modelling framework to quantify the effects of passaging cell lines

In vitro cell culture is routinely used to grow and supply a sufficiently large number of cells for various types of cell biology experiments. Previous experimental studies report that cell characteristics evolve as the passage number increases, and various cell lines can behave differently at high passage numbers. To provide insight into the putative mechanisms that might give rise to these differences, we perform in silico experiments using a random walk model to mimic the in vitro cell culture process. Our results show that it is possible for the average proliferation rate to either increase or decrease as the passaging process takes place, and this is due to a competition between the initial heterogeneity and the degree to which passaging damages the cells. We also simulate a suite of scratch assays with cells from near-homogeneous and heterogeneous cell lines, at both high and low passage numbers. Although it is common in the literature to report experimental results without disclosing the passage number, our results show that we obtain significantly different closure rates when performing in silico scratch assays using cells with different passage numbers. Therefore, we suggest that the passage number should always be reported to ensure that the experiment is as reproducible as possible. Furthermore, our modelling also suggests some avenues for further experimental examination that could be used to validate or refine our simulation results.

biophysics↗

bioRxiv2017

Traction force screening enabled by compliant PDMS elastomers

Acto-myosin contractility is an essential element of many aspects of cellular biology, and manifests as traction forces that cells exert on their surroundings. The central role of these forces makes them a novel principal therapeutic target in diverse diseases. This requires accurate and higher capacity measurements of traction forces; however, existing methods are largely low throughput, limiting their utility in broader applications. To address this need, we employ Fourier-transform traction force microscopy in a parallelized 96-well format, which we refer to as contractile force screening (CFS).Critically, rather than the frequently employed hydrogel polyacrylamide (PAA), we fabricate these plates using polydimethylsiloxane (PDMS) rubber. Key to this approach is that the PDMS used is very compliant, with a lower-bound Youngs modulus of approximately 0.7 kPa. We subdivide these monolithic substrates spatially into biochemically independent wells, creating a uniform multiwell platform for traction force screening. We demonstrate the utility and versatility of this platform by quantifying the compound and dose-dependent contractility responses of human airway smooth muscle cells and retinal pigment epithelial cells.

biophysics↗

bioRxiv2017

Immature HIV-1 Lattice Assembly Dynamics are Regulated by Scaffolding from Nucleic Acid and the Plasma Membrane

The packaging and budding of Gag polyprotein and viral ribonucleic acid (RNA) is a critical step in the human immunodeficiency virus-1 (HIV-1) lifecycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from sub-nanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA-SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while over-expression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.\n\nSIGNIFICANCE STATEMENTIn order for human immunodeficiency virus to proliferate, viral proteins and genomic dimers are assembled at host cell membranes and released as immature virions. Disrupting this key intermediate step in viral replication is a potential target for treatment. However, a detailed molecular view of this process remains lacking. Here, we elucidate a network of constitutive interactions that regulate viral assembly dynamics through a combined computational and experimental approach. Specifically, our analysis reveals the active roles of nucleic acid and the membrane as scaffolds that promote the multimerization of Gag polyprotein which proceeds through multi-step and self-correcting nucleation. Our findings also illustrate the functional importance of the N-terminal, C-terminal, and spacer peptide 1 protein domains.

biophysics↗

bioRxiv2017

Mechanism of genome interrogation: How CRISPR RNA-guided Cas9 proteins locate specific targets on DNA

The ability to precisely edit and modify genome opens endless opportunities to investigate fundamental properties of living systems as well as to advance various medical techniques and bioengineering applications. This possibility is now close to reality due to a recent discovery of the adaptive bacterial immune system, which is based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) that utilize RNA to find and cut the double-stranded DNA molecules at specific locations. Here we develop a quantitative theoretical approach to analyze the mechanism of target search on DNA by CRISPR RNA-guided Cas9 proteins, which is followed by a selective cleavage of nucleic acids. It is based on a discrete-state stochastic model that takes into account the most relevant physical-chemical processes in the system. Using a method of first-passage processes, a full dynamic description of the target search is presented. It is found that the location of specific sites on DNA by CRISPR Cas9 proteins is governed by binding first to protospacer adjacent motif (PAM) sequences on DNA, which is followed by reversible transitions into DNA interrogation states. In addition, the search dynamics is strongly influenced by the off-target cutting. Our theoretical calculations allows us to explain the experimental observations and to give experimentally testable predictions. Thus, the presented theoretical model clarifies some molecular aspects of the genome interrogation by CRISPR RNA-guided Cas9 proteins.\n\nInsert Received for publication Date and in final form Date.

biophysics↗

bioRxiv2017

HtrA1 activation is driven by an allosteric mechanism of inter-monomer communication

The human protease family HtrA is responsible for preventing protein misfolding and mislocalization, and a key player in several cellular processes. Among these, HtrA1 is implicated in several cancers, cerebrovascular disease and age-related macular degeneration. HtrA1 activation, although very relevant for drug-targeting this protease, remains poorly characterized. Our work provides a mechanistic step-by-step description of HtrA1 activation and regulation. We report that the HtrA1 trimer is regulated by an allosteric mechanism by which monomers relay the activation signal to each other, in a PDZ-domain independent fashion. Notably, we show that inhibitor binding is precluded if HtrA1 monomers cannot communicate with each other. Our study establishes how HtrA1 oligomerization plays a fundamental role in proteolytic activity. Moreover, it offers a structural explanation for HtrA1-defective pathologies as well as mechanistic insights into the degradation of complex extracellular fibrils such as tubulin, amyloid beta and tau that belong to the repertoire of HtrA1.\n\nHighlightsO_LIMonomeric HtrA1 is activated by a gating mechanism.\nC_LIO_LITrimeric HtrA1 is regulated by PDZ-independent allosteric monomer cross-talk.\nC_LIO_LIHtrA1 oligomerization is key for proteolytic activity.\nC_LIO_LISubstrate-binding is precluded if monomers cannot communicate with each other.\nC_LI

biophysics↗

bioRxiv2017

Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

biophysics↗

bioRxiv2017

OPUS-CSF: A C-atom-based Scoring Function for Ranking Protein Structural Models

We report a C-atom-based scoring function, named OPUS-CSF, for ranking protein structural models. Rather than using traditional Boltzmann formula, we built a scoring function (CSF score) based on the native distributions (analyzed through entire PDB) of coordinate components of mainchain C atoms on selected residues of peptide segments of 5, 7, 9, and 11 residues in length. In testing OPUS-CSF on decoy recognition, it maximally recognized 257 native structures out of 278 targets in 11 commonly used decoy sets, significantly more than other popular all-atom empirical potentials. The average correlation coefficient with TM-score was also comparable with those of other potentials. OPUS-CSF is a highly coarse-grained scoring function, which only requires input of partial mainchain information, and very fast. Thus it is suitable for applications at early stage of structural building.

biophysics↗

bioRxiv2017

Dependence of Intravoxel Incoherent Motion MR threshold b-value selection for separating perfusion and diffusion compartment and liver fibrosis diagnostic performance.

PurposeTo explore how the selection of threshold b-value impacts Intravoxel Incoherent Motion (IVIM) diffusion parameters of PF (f), Dslow (D),and Dfast (D*) values and their performance for liver fibrosis detection.\n\nMaterials and MethodsFifteen healthy volunteers and 33 hepatitis-b patients were included. With a 1.5 T MR scanner and respiration gating, IVIM data was acquired with 10 b-values of 10,20,40,60,80,100,150, 200, 400, and 800 s/mm2. Signal measurement was performed on right liver. Segmented-unconstrained analysis was used to compute IVIM parameters, and six threshold b-values between 40 and 200 s/mm2 were compared. PF, Dslow, and Dfast values were placed along the x-axis, y-axis, and z-axis, and a plane was defined to separate volunteers from patients.\n\nResultsHigher threshold b-values were associated with higher PF measurement; while lower threshold b-values led to higher Dslow and Dfast measurements. The dependence of PF, Dslow, and Dfast on threshold b-value differed between healthy livers and fibrotic livers; with the healthy livers showing a higher dependence. Threshold b-value=60 s/mm2 showed the largest mean distance between healthy liver datapoints vs. fibrotic liver datapoints in 3-dimensional space.\n\nConclusionFor segmented-unconstrained analysis, the selection of threshold b-value=60 s/mm2 improves IVIM diffusion differentiation between healthy livers and fibrotic livers.

biophysics↗

bioRxiv2017

Cytosolic proteins can exploit membrane localization to trigger functional assembly

Cell division, endocytosis, and viral budding would not function without the localization and assembly of protein complexes on membranes. What is poorly appreciated, however, is that by localizing to membranes, proteins search in a reduced space that effectively drives up concentration. Here we derive an accurate and practical analytical theory to quantify the significance of this dimensionality reduction in regulating protein assembly on membranes. We define a simple metric, an effective equilibrium constant, that allows for quantitative comparison of protein-protein interactions with and without membrane present. To test the importance of membrane localization for driving protein assembly, we collected the protein-protein and protein-lipid affinities, protein and lipid concentrations, and volume-to-surface-area ratios for 46 interactions between 37 membrane-targeting proteins in human and yeast cells. We find that many of the protein-protein interactions between pairs of proteins involved in clathrin-mediated endocytosis in human and yeast cells can experience enormous increases in effective protein-protein affinity (10-1000 fold) due to membrane localization. Localization of binding partners thus triggers robust protein complexation, suggesting that it can play an important role in controlling the timing of endocytic protein coat formation. Our analysis shows that several other proteins involved in membrane remodeling at various organelles have similar potential to exploit localization. The theory highlights the master role of phosphoinositide lipid concentration, the volume-to-surface-area ratio, and the ratio of 3D to 2D equilibrium constants in triggering (or preventing) constitutive assembly on membranes. Our simple model provides a novel quantitative framework for interpreting or designing in vitro experiments of protein complexation influenced by membrane binding.

biophysics↗

bioRxiv2017

Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA and actin filaments

We used fluorescence spectroscopy and electron microscopy to determine how binding of ATP, nucleation-promoting factors (NPF), actin monomers and actin filaments change the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C-termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Forster resonance energy transfer (FRET) efficiency (ETeff) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide binding clefts of the Arp subunits. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ETeff between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected a different structural change that involves bringing ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. Each of the ligands that activates Arp2/3 complex changes the structure in different ways, each leading progressively to fully activated Arp2/3 complex on the side of a filament.

biophysics↗

bioRxiv2017

Collective membrane dynamics emerging from curvature-dependent spatial coupling

Membrane curvature has been recognized as an active participant of fundamental biological processes including vesicular transport and organelle biogenesis, but its effects on membrane remodeling are typically local. Here we show membrane curvature plays a critical role in propagating cortical waves and modulating mesoscale dynamics in living cells. We employ a membrane shape-dependent mechanochemical feedback model to account for the observed oscillatory travelling waves of Cdc42, F-BAR proteins and actin. We demonstrate that oscillatory membrane shape changes accompany and are required for such spatiotemporal patterns. In addition, modulating the curvature preference of the F-BAR proteins or membrane tension perturbs wave propagation. Our findings identify a distinct role of membrane curvature in mediating collective dynamics of cortical proteins and provide a molecular framework for integrating membrane mechanics and biochemical signaling in the context of subcellular pattern formation.

biophysics↗