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bioRxiv2022

Changing flows balance nutrient absorption and bacterial growth along the gut

Small intestine motility and its ensuing flow of luminal content impact both nutrient absorption and bacterial growth. To explore this interdependence we introduce a biophysical description of intestinal flow and absorption. Rooted in observations of mice we identify the average flow velocity as the key control of absorption efficiency and bacterial growth, independently of the exact contraction pattern. We uncover self-regulation of contraction and flow in response to nutrients and bacterial levels to promote efficient absorption while restraining detrimental bacterial overgrowth.

biophysics↗

bioRxiv2022

Structural and functional determinants inferred from deep mutational scans

Mutations that affect protein binding to a cognate partner primarily occur either at buried residues or at exposed residues directly involved in partner binding. Distinguishing between these two categories based solely on mutational phenotypes is challenging. The bacterial toxin CcdB kills cells by binding to DNA Gyrase. Cell death is prevented by binding to its cognate antitoxin CcdA, at an extended interface that partially overlaps with the GyrA binding site. Using the CcdAB toxin-antitoxin (TA) system as a model, a comprehensive site-saturation mutagenesis library of CcdB was generated in its native operonic context. The mutational sensitivity of each mutant was estimated by evaluating the relative abundance of each mutant in two strains, one resistant and the other sensitive to the toxic activity of the CcdB toxin, through deep sequencing. The ability to bind CcdA was inferred through a RelE reporter gene assay, since the CcdAB complex binds to its own promoter, repressing transcription. By analysing mutant phenotypes in the CcdB sensitive, CcdB resistant and RelE reporter strains, it was possible to assign residues to buried, CcdA interacting or GyrA interacting sites. A few mutants were individually constructed, expressed, and biophysically characterised to validate molecular mechanisms responsible for the observed phenotypes. Residues inferred to be important for antitoxin binding, are also likely to be important for rejuvenating CcdB from the CcdB-Gyrase complex. Therefore, even in the absence of structural information, when coupled to appropriate genetic screens, such high-throughput strategies can be deployed for predicting structural and functional determinants of proteins. Broader Impact StatementPartial loss-of-function mutations predominantly occur either at buried-site or exposed, active-site residues. We report a facile method to identify multiple binding sites for different interacting partners for a protein, and distinguish them from buried site and exposed non active-site residues, solely from mutational data.

biophysics↗

bioRxiv2022

Tween-20 induces the structural remodelling of single lipid vesicles

The interaction of Tween-20 with lipid membranes is crucial for a number of biotechnological applications including viral inactivation and membrane protein extraction, but the underlying mechanistic details have remained elusive. Evidence from ensemble assays supports a global model of Tween-20 induced membrane disruption that broadly encompasses association of the surfactant with the membrane surface, membrane fragmentation and the release of mixed micelles to solution, but whether this process involves intermediate and dynamic transitions between regimes is an open question. In search of the mechanistic origins of membrane disruption, increasing focus is put on identifying Tween-20 interactions with highly controllable model membranes. In light of this, and to unveil quantitative mechanistic details, we employed highly interdisciplinary biophysical approaches, including quartz-crystal microbalance with dissipation monitoring, steady-state and time-resolved fluorescence and FRET spectroscopy, dynamic light scattering, fluorescence correlation spectroscopy, wide-field single-vesicle imaging and scanning electron microscopy, to interrogate the interactions between Tween-20 and both freely-diffusing and surface-immobilized model-membrane vesicles. Using ultrasensitive sensing approaches, we discovered that Tween-20 leads to a stepwise and phase-dependent structural remodelling of sub-micron sized vesicles that includes permeabilization and swelling, even at detergent concentrations below the critical micellar concentration. These insights into the structural perturbation of lipid vesicles upon Tween-20 interaction highlight the impact on vesicle conformation prior to complete solubilization, and the tools presented may have general relevance for probing the interaction between lipid vesicles and a wide variety of disruptive agents.

biophysics↗

bioRxiv2022

Integrative experimental/computational approach establishes active cellular protrusion as the primary driving force of phagocytic spreading by immune cells

The dynamic interplay between cell adhesion and protrusion is a critical determinant of many forms of cell motility. When modeling cell spreading on adhesive surfaces, traditional mathematical treatments often consider passive cell adhesion as the primary, if not exclusive, mechanistic driving force of this cellular motion. To better assess the contribution of active cytoskeletal protrusion to immune-cell spreading during phagocytosis, we here develop a computational framework that allows us to optionally investigate both purely adhesive spreading ("Brownian zipper hypothesis") as well as protrusion-dominated spreading ("protrusive zipper hypothesis"). We model the cell as an axisymmetric body of highly viscous fluid surrounded by a cortex with uniform surface tension and incorporate as potential driving forces of cell spreading an attractive stress due to receptor-ligand binding and an outward normal stress representing cytoskeletal protrusion, both acting on the cell boundary. We leverage various model predictions against the results of a directly related experimental companion study of human neutrophil phagocytic spreading on substrates coated with different densities of antibodies. We find that the concept of adhesion-driven spreading is incompatible with experimental results such as the independence of the cell-spreading speed on the density of immobilized antibodies. In contrast, the protrusive zipper model agrees well with experimental findings and, when adapted to simulate cell spreading on discrete adhesion sites, it also reproduces the observed positive correlation between antibody density and maximum cell-substrate contact area. Together, our integrative experimental/computational approach shows that phagocytic spreading is driven by cellular protrusion, and that the extent of spreading is limited by the density of adhesion sites. Author SummaryTo accomplish many routine biological tasks, cells must rapidly spread over different types of surfaces. Here, we examine the biophysical underpinnings of immune cell spreading during phagocytosis, the process by which white blood cells such as neutrophils engulf pathogens or other foreign objects. Our computational framework models the case in which a human neutrophil spreads over a flat surface coated with antibodies, which we also test experimentally in a companion paper. Our primary purpose is to assess whether phagocytic spreading is actively driven by protrusive forces exerted by the cell, or passively by adhesive forces acting between receptors in the cell membrane and antibodies on the surface. By directly comparing our model predictions to experimental results, we demonstrate that phagocytic spreading is primarily driven by protrusion, but the extent of spreading is still limited by the availability of binding sites. Our findings improve the fundamental understanding of phagocytosis and may also pave the way for future investigations of the balance between adhesion and protrusion in other forms of cell spreading, such as wound healing or cancer cell migration.

biophysics↗

bioRxiv2022

Efficient quantification of lipid packing defect sensing by amphipathic peptides; comparing Martini 2 & 3 with CHARMM36

In biological systems, proteins can be attracted to curved or stretched regions of lipid bilayers by sensing hydrophobic defects in the lipid packing on the membrane surface. Here, we present an efficient end-state free energy calculation method to quantify such sensing in molecular dynamics simulations. We illustrate that lipid packing defect sensing can be defined as the difference in mechanical work required to stretch a membrane with and without a peptide bound to the surface. We also demonstrate that a peptides ability to concurrently induce excess leaflet area (tension) and elastic softening - a property we call the characteristic area of sensing (CHAOS) - and lipid packing sensing behavior are in fact two sides of the same coin. In essence, defect sensing displays a peptides propensity to generate tension. The here-proposed mechanical pathway is equally accurate yet, computationally, about 40 times less costly than the commonly used alchemical pathway (thermodynamic integration), allowing for more feasible free energy calculations in atomistic simulations. This enabled us to directly compare the Martini 2 and 3 coarse-grained and the CHARMM36 atomistic force-fields in terms of relative binding free energies for six representative peptides including the curvature sensor ALPS and two antiviral amphipathic helices (AH). We observed that Martini 3 qualitatively reproduces experimental trends, whilst producing substantially lower (relative) binding free energies and shallower membrane insertion depths compared to atomistic simulations. In contrast, Martini 2 tends to overestimate (relative) binding free energies. Finally, we offer a glimpse into how our end-state based free energy method can enable the inverse design of optimal lipid packing defect sensing peptides when used in conjunction with our recently developed Evolutionary Molecular Dynamics (Evo-MD) method. We argue that these optimized defect sensors - aside from their biomedical and biophysical relevance - can provide valuable targets for the development of lipid force-fields. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/482978v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1872c25org.highwire.dtl.DTLVardef@1635f0eorg.highwire.dtl.DTLVardef@f5ad91org.highwire.dtl.DTLVardef@156209f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Theoretical Quantification of the Polyvalent Binding of Nanoparticles Coated with Peptide-MHC to TCR-Nanoclusters

Nanoparticles (NPs) coated with pMHCs can reprogram a specific type of CD4+ T cells into diseasesuppressing T regulatory type 1 cells by binding to their TCRs expressed as TCR-nanoclusters (TCRnc). NP size and number of pMHCs coated on them (called valence) can be adjusted to increase their efficacy. Here we explore how this polyvalent interaction is manifested and examine if it can facilitate T cell activation. This is done by developing a multiscale biophysical model that takes into account the complexity of this interaction. Using the model, we quantify pMHC insertion probabilities, dwell time of NP binding, TCRnc carrying capacity, the distribution of covered and bound TCRs by NPs, and cooperativity in the binding of pMHCs within the contact area. Model fitting and parameter sweeping further reveal that moderate jumps between IFN{gamma} dose-response curves at low valences can occur, suggesting that the geometry of NP binding can prime T cells for activation.

biophysics↗

bioRxiv2022

Inherent dynamics of maltose binding protein (MBP) are immune to the native environment.

Biophysical characterizations of proteins typically rely on a reductionistic approach, studying proteins in a highly purified from and in absence of their natural cellular environment. Little is known about how the highly crowded conditions prevalent within living cells influence the dynamic structures proteins on the molecular level. To address this outstanding question, we characterize here the dynamic behavior of the periplasmic model protein MBP from Escherichia coli in situ, confined in the native lumen of bacterial outer membrane vesicles. To this end we determine the dynamics of side-chain methyl groups of MBP across several timescales and compare them to purified in vitro MBP. We find that the inherent dynamics of MBP are surprisingly insensitive to the native cellular environment and that the molecular motion of the protein is mainly impacted on a global level.

biophysics↗

bioRxiv2022

Three-dimensional structure of the single domain cupredoxin AcoP

Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 [A] resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network.

biophysics↗

bioRxiv2022

Nanomechanical and microrheological properties of bladder cancer cells at cellular and spheroid levels

Cancer progression is associated with changes in cell mechanical and rheological properties that could be probed by atomic force microscopy (AFM). In this study, we applied AFM to measure elastic (by compressing the cells) and viscoelastic (by applying shear stress) properties of bladder cancers in relation to their culture morphology (in single cells, cell monolayers, and spheroids). Three different cell lines, HCV29 (non-malignant cell cancer of ureter), HT1376 (grade III bladder carcinoma), T24 (grade IV transitional cell carcinoma), were investigated. Nanoindentation measurements only differentiate between non-malignant and cancer cells, but it is difficult to distinguish between specific bladder cancers. By applying microrheological measurements, we confirm that non-malignant cells are more rigid than cancer cells but more importantly, it was possible to differentiate between two cancerous cell lines, regardless of the culture conditions. As each of them is characterized by a distinct actin filament network inside the cell, we showed that actin filaments are a key element in defining the rheological properties of spheroids originating from cells having thick actin bundles. Our results showed that HCV29 cells are more rigid than the studied cancer cells, indicating that normal cells are resistant to compressive and shear forces. Therefore, we conclude that cell mechanical and rheological properties may serve as a biophysical marker to distinguish normal and cancer cells of different malignancies.

biophysics↗

bioRxiv2022

Pinning transition in biofilm structure driven by active layer dynamics

Surface-attached communities of microbes, known as biofilms, are diverse in their morphologies. Characterising distinct types of biofilm spatial structure, and understanding how they emerge, can shed light on the fundamental biological and biophysical mechanisms involved, and can improve our understanding of evolution in biofilms. Here, we perform long-time individual-based simulations of growing biofilms. We observe distinct types of biofilm spatial structure depending on the parameters, and we classify these into three phases according to the behaviour of the active layer of growing cells at the biofilm interface. In the unpinned phase, the biofilm is smooth and the active layer is unbroken with no gaps. In the transiently pinned phase, short-lived gaps in the active layer arise, which can cause local parts of the biofilm interface to pin, or become stationary relative to the moving front. In the pinned phase these pinning sites persist, leading to fingering of the biofilm interface. We show that pinning arises due to the dynamical behaviour of active layer gaps, and observe that the relative magnitudes of the active layer thickness and the active layer fluctuations are important in this process. We demonstrate a direct connection between biofilm pinning and interface roughness, and we show that the pinning phase transition is well described by a control parameter that combines the average and standard deviation of the active layer thickness. Taken together, our work suggests a role for active layer dynamics in controlling pinning of the biofilm interface and hence biofilm morphology.

biophysics↗

bioRxiv2022

Model biomolecular condensates have heterogeneous structure quantitatively dependent on the interaction profile of their constituent macromolecules

Biomolecular condensates play numerous roles in cells by selectively concentrating client proteins while excluding others. These functions are likely to be sensitive to the spatial organization of the scaffold proteins forming the condensate. We use coarse-grained molecular simulations to show that model intrinsically-disordered proteins phase separate into a heterogeneous, structured fluid characterized by a well-defined length scale. The proteins are modelled as semi-flexible polymers with punctate, multifunctional binding sites in good solvent conditions. Their dense phase is highly solvated with a spatial structure that is more sensitive to the separation of the binding sites than their affinity. We introduce graph theoretic measures to show that the proteins are heterogeneously distributed throughout the dense phase, an effect that increases with increasing binding site number, and exhibit multi-timescale dynamics. The simulations predict that the structure of the dense phase is modulated by the location and affinity of binding sites distant from the termini of the proteins, while sites near the termini more strongly affect its phase behaviour. The relations uncovered between the arrangement of weak interaction sites on disordered proteins and the material properties of their dense phase can be experimentally tested to give insight into the biophysical properties and rational design of biomolecular condensates.

biophysics↗

bioRxiv2022

Extracellular vesicle fusion visualized by cryo-EM

Extracellular vesicles (EVs) transfer bioactive molecules between cells in a process reminiscent of enveloped viruses. EV cargo delivery is thought to occur by protein-mediated and pH-dependent membrane fusion of the EV and the cellular membrane. However, there is a lack of methods to identify the fusion proteins and resolve their mechanism. We developed and benchmarked an in vitro biophysical assay to investigate EV membrane fusion. The assay was standardized by directly comparing EV- and viral-fusion with liposomes. We show that EVs and retroviruses fuse with liposomes mimicking the membrane composition of the late endosome in a pH and protein-dependent manner. Moreover, we directly visualize the stages of membrane fusion using cryo-electron tomography. We find that, unlike most retroviruses, EVs remain fusogenic after acidification and re-neutralization. These results provide novel insights into the EV cargo delivery mechanism and an experimental approach to identify the EV fusion machinery.

biophysics↗

bioRxiv2022

Growth instabilities shape morphology and genetic diversity of cellular aggregates

Cellular aggregates assume an incredible variety of shapes ranging from circular molds to irregular tumors. While we understand many of the mechanisms responsible for these spatial patterns, little is known about how the shape of an aggregate influences its ecology and evolution. Here, we investigate this relationship in the context of microbial colonies grown on hard agar plates. This a well-studied system that exhibits a transition from smooth circular disks to more irregular and rugged shapes as either the nutrient concentration or cellular motility is decreased. Starting from a mechanistic model of colony growth, we identify two dimensionless quantities that determine how morphology and genetic diversity of the population depend on the model parameters. Our simulations further reveal that population dynamics cannot be accurately described by the commonly-used surface growth models. Instead, one has to explicitly account for the emergent growth instabilities and demographic fluctuations. Overall, our work links together environmental conditions, colony morphology, and evolution. This link is essential for a rational design of concrete, biophysical perturbations to steer evolution in the desired direction.

biophysics↗

bioRxiv2022

Reactive oxygen species generated by infrared laser light in optical tweezers inhibits the germination of bacterial spores

Bacterial spores are highly resistant to heat, radiation, and various disinfection chemicals. These impacts on the biophysical and physicochemical properties of spores can be studied on the single-cell level using optical tweezers. However, the effect of the trapping laser on spores germination rate is not fully understood. In this work, we assess the impact of 1064 nm laser light on the germination of Bacillus thuringiensis spores. The results show that the germination rate of spores after laser exposure follows a sigmoid dose-response relationship, with only 15 % of spores germinating after 20 J of laser light. Under anaerobic growth conditions, the percentage of germinating spores at 20 J increased to 65 %. The results thereby indicate that molecular oxygen is a major contributor to the germination-inhibiting effect observed. Thus, our study highlights the risk for optical trapping of spores and ways to mitigate it.

biophysics↗

bioRxiv2022

Scanning Single Molecule Localization Microscopy (scanSMLM) for super-resolution optical volume imaging

Over the last decade, single molecule localization microscopy (SMLM) has developed into a set of powerful techniques that has improved spatial resolution over diffraction-limited microscopy and demonstrated the ability to resolve biological features at the very molecular scale. We introduce a single molecule based scanning SMLM (scanSMLM) system that enables rapid volume imaging. Using a standard widefield illumination, the system employs a scanning based detection 4f-sub-system suited for volume interrogation. The 4f system comprises of a combination of electrically-tunable lens and high NA detection objective lens. By rapidly changing the aperture (or equivalently the focus) of electrically-tunable lens (ETL) in a 4f detection system, the selectivity of axial (Z) plane can be achieved in the object plane, for which the corresponding image forms in the image/detector plane. So, in-principle one can scan the object volume by just changing the aperture of ETL. To carry out volume imaging, a cyclic scanning scheme is developed and compared with conventional scanning routinely used in SMLM. The scanning scheme serves the purpose of distributing photobleaching evenly by ensuring uniform dwell time on each frame for collecting data (single molecule events) throughout the specimen volume. With minimal change in the system hardware (requiring an addition of ETL lens and related hardware for step-voltage generation) in the existing SMLM system, volume scanning (along z-axis) can be achieved. To demonstrate, we imaged fluorescent beads embedded in a gel-matrix 3D block as a test sample. Subsequently, scanSMLM is employed to understand clustering of HA single molecules in a transfected cell (Influenza A disease model). The system for the first time enables visualization of HA distribution in a 3D cells that reveal its clustering across the cell volume. Critical biophysical parameters related to HA clusters (density, #HA/cluster and clustered fraction) are also determined.

biophysics↗

bioRxiv2022

Thermal Effects on Neurons During Stimulation of the Brain

All artificial stimulation of the brain deposits thermal energy in the brain. This occurs through either Joule heating of the conductors carrying current through electrodes and magnetic coils, or through dissipation of energy in the conductive brain. Similarly, temperature affects all biological processes and chemical reactions. Although electrical interaction with brain tissue is inseparable from thermal effects when electrodes are used, magnetic induction enables us to separate Joule heating from induction effects by contrasting AC and DC driving of magnetic coils using the same energy deposition within the conductors. Since mammalian cortical neurons have no known sensitivity to static magnetic fields, and if there is no evidence of effect on spike timing to oscillating magnetic fields, we can presume that the induced electrical currents within the brain are below the molecular shot noise where any interaction with tissue is purely thermal. In this study, we examined a range of frequencies produced from micromagnetic coils operating below the molecular shot noise threshold for electrical interaction with single neurons. We found that small temperature increases and decreases of 1{degrees}C caused consistent transient suppression and excitation of neurons during temperature change. Numerical modeling of the biophysics demonstrated that the Na-K pump, and to a lesser extent the Nernst potential, could account for these transient effects. Such effects are dependent upon compartmental ion fluxes, and the rate of temperature change. A new bifurcation is described in the model dynamics that accounts for the transient suppression and excitation; in addition, we note the remarkable similarity of this bifurcations rate dependency with other thermal rate-dependent tipping points in planetary warming dynamics. Furthermore, bifurcations in the steady state dynamics leading to stable firing suppression are described for slightly higher temperatures. These experimental and theoretical findings demonstrate that stimulation of the brain must take into account small thermal effects that are ubiquitously present in electrical and magnetic stimulation. More sophisticated models of electrical current interaction with neurons combined with thermal effects will be required in order to more accurately enable model-based control of neuronal circuitry.

biophysics↗

bioRxiv2022

The purified recombinant FAT domain of the Focal Adhesion Kinase does not bind directly to recombinant Talin or MBD2 in vitro

Controlled localization and activation of the focal adhesion kinase (FAK) functionally links adhesion, migration and survival of the cell. The C-terminal focal adhesion targeting (FAT) domain of FAK is an important regulator of the localization, activation and molecular associations of FAK. Here, we aimed to investigate the structural basis for how FAK FAT binds to Talin and MBD2, which were previously reported to be cytoplasmic and nuclear ligands, respectively. Using several biophysical methods with purified recombinantly expressed protein constructs, we failed to observe measurable interactions between FAT and either the Talin FERM domain or MBD2. We conclude that the association of FAT with these proteins requires additional factors or post-translational modifications not present in bacterially produced purified proteins.

biophysics↗

bioRxiv2022

Thermophoresis of Molecules and Structures of different Sizes in Self-assembled Biomatrices

Upon subjecting biomolecules to non-equilibrium conditions, many biochemical and biophysical features such as biomolecular diffusion, protein folding, interaction kinetics, as well as enzyme-catalyzed reactions can be characterized in an aqueous solution. However, most assays under non-equilibrium conditions cannot be performed in complex self-assembled biomatrices (e.g. extracellular matrices) due to the limitations associated with sample handling, reaction design, and optical detection. Herein, we report the study of biomolecular thermodiffusion in non-covalently assembled synthetic or naturally derived hydrogels. This approach has been demonstrated with a large variety of analytes, including small molecules, polysaccharides, DNAs, DNA origami, and proteins in various polymer networks. The in-biomatrix method has also shown advantages over in-solution measurements: First, it allows us to analyze biomolecules in 3D matrices in a high-throughput fashion. Second, the aggregation of analytes can be remarkably prevented. Although the underlying physics of thermodiffusion is still not well-understood, we demonstrated that the thermodiffusion of surrounding networks will enhance the thermodiffusion of the analyte, an effect counteracting the hindered movement by the polymer network.

biophysics↗