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bioRxiv2024

A possible path to persistent re-entry waves at the outlet of the left pulmonary vein

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia, often evolving from paroxysmal episodes to persistent stages over an extended timeframe. While various factors contribute to this progression, the precise biophysical mechanisms driving it remain unclear. Here we explore how rapid firing of cardiomyocytes at the outlet of the pulmonary vein of the left atria can create a substrate for a persistent re-entry wave. This is grounded in a recently formulated mathematical model of the regulation of calcium ion channel density by intracellular calcium concentrations. According to the model, the density of membrane proteins carrying calcium ions is controlled by the intracellular calcium concentrations. In particular, if the concentration increases above a certain target level, the calcium current is weakened in order to restore the target level of calcium. During rapid pacing, the intracellular calcium concentration of the cardiomyocytes increases leading to a substantial reduction of the calcium current across the membrane of the myocytes, which again reduces the action potential duration. In a spatially resolved cell-based model of the outlet of the pulmonary vein of the left atria, we show that the reduced action potential duration can lead to re-entry. Initiated by rapid pacing, often stemming from paroxysmal AF episodes lasting several days, the reduction in calcium current is a critical factor. Our findings illustrate how such episodes can foster a conducive environment for persistent AF through electrical remodeling, characterized by diminished calcium currents. This underscores the importance of promptly addressing early AF episodes to prevent their progression to chronic stages.

biophysics↗

bioRxiv2024

Conditions for the co-existence of promoter and gene-body condensates

In cells, transcription is tightly regulated on multiple layers. The condensation of the transcription machinery into distinct phases is hypothesised to spatio-temporally fine tune RNA polymerase II behaviour during two key stages, transcription initiation and the elongation of the nascent RNA transcripts. However, it has remained unclear whether these phases would mix when present at the same time or remain distinct chemical environments; either as multi-phase condensates or by forming entirely separate condensates. Here we combine particle-based multi-scale simulations and experiments in the model organism C. elegans to characterise the biophysical properties of RNA polymerase II condensates. Both simulations and the in vivo work describe a lower critical solution temperature (LCST) behaviour of RNA Polymerase II, with condensates dissolving at lower temperatures whereas higher temperatures promote condensate stability. Importantly this gradual change in temperature correlates with an incremental transcriptional response to temperature, but is largely uncoupled from the classical stress response. The LCST behaviour of CTD also highlights that these condensates are physio-chemically distinct from heterochromatin condensates. Expanding the simulations we model how the degree of phosphorylation of the disordered C-terminal domain of RNA polymerase II (CTD), which is characteristic for each step of transcription, controls demixing of CTD and pCTD in line with phase separation experiments. We show that the two phases putatively underpinning the initiation of transcription and transcription elongation constitute distinct chemical environments and are in agreement with RNA polymerase II condensates observed in C. elegans embryos by super resolution microscopy. Our analysis reveals how depending on its post-translational modifications and its interaction partners a single protein can adopt multiple morphologies and how partially engulfed condensates promote the selective recruitment of additional factors to the different phases.

biophysics↗

bioRxiv2024

Transcription factors ERα and Sox2 have differing multiphasic DNA and RNA binding mechanisms

Many transcription factors (TFs) have been shown to bind RNA, leading to open questions regarding the mechanism(s) of this RNA binding and its role in regulating TF activities. Here we use biophysical assays to interrogate the kon, koff, and Kd for DNA and RNA binding of two model human transcription factors, ER and Sox2. Unexpectedly, we found that both proteins exhibited multiphasic nucleic acid binding kinetics. We propose that Sox2 RNA and DNA multiphasic binding kinetics could be explained by a conventional model for sequential Sox2 monomer association and dissociation. In contrast, ER nucleic acid binding exhibited biphasic dissociation paired with novel triphasic association behavior, where two apparent binding transitions are separated by a 10-20 min "lag" phase depending on protein concentration. We considered several conventional models for the observed kinetic behavior, none of which adequately explained all the ER nucleic acid binding data. Instead, simulations with a model incorporating sequential ER monomer association, ER nucleic acid complex isomerization, and product "feedback" on isomerization rate recapitulated the general kinetic trends for both ER DNA and RNA binding. Collectively, our findings reveal that Sox2 and ER bind RNA and DNA with previously unappreciated multiphasic binding kinetics, and that their reaction mechanisms differ with ER binding nucleic acids via a novel reaction mechanism.

biophysics↗

bioRxiv2024

Energy landscapes of peptide-MHC binding

Molecules of the Major Histocompatibility Complex (MHC) present short protein fragments peptides on the cell surface, an important step in T cell immune recognition. MHC-I molecules process peptides from intracellular proteins; MHC-II molecules act in antigen-presenting cells and present peptides derived from extracellular proteins. Here we show that the sequence-dependent energy landscapes of MHC-peptide binding encode class-specific nonlinearities (epistasis). MHC-I has a smooth landscape with global epistasis; the binding energy is a simple deformation of an underlying linear trait. This form of epistasis enhances the discrimination between strong-binding peptides. In contrast, MHC-II has a rugged landscape with idiosyncratic epistasis: binding depends on detailed amino acid combinations at multiple positions of the peptide sequence. The form of epistasis affects the learning of energy landscapes from training data. For MHC-I, a low-complexity problem, we derive a simple matrix model of binding energies that outperforms current models trained by machine learning. For MHC-II, higher complexity prevents learning by simple regression methods. Epistasis also affects the energy and fitness effects of mutations in antigen-derived peptides (epitopes). In MHC-I, large-effect mutations occur predominantly in anchor positions of strong-binding epitopes. In MHC-II, large effects depend on the background epitope sequence but are broadly distributed over the epitope, generating a bigger target for escape mutations from T cell immunity than for MHC-I. Author SummaryT cell immunity involves the binding of short peptides to the intracellular MHC recognition machinery. Understanding how the binding energy depends on the peptide sequence is key to computationally predict immune recognition and immune escape evolution, for example, of pathogens and cancer cells. We find nonlinear energy landscapes that depend on the recognition pathway: smooth and easy to learn for MHC class I, rugged and difficult to learn for class II. Together, this work establishes links between biophysical origin, nonlinear structure, learnability from data, and biological implications for protein interaction landscapes.

biophysics↗

bioRxiv2024

A realistic algorithm for microtubule-based nucleation reveals large sensitivity to cell geometry of the plant cortical array

Many plant cell functions, including cell morphogenesis and anisotropic growth, rely on the self-organisation of cortical microtubules into aligned arrays with the correct orientation. An important ongoing debate is how cell geometry, wall mechanical stresses, and other internal and external cues are integrated to determine the orientation of the cortical array. Here, we demonstrate that microtubule-based nucleation can markedly shift the balance between these often competing directional cues. For this, we developed a novel, more realistic model for microtubule-based nucleation in the simulation platform CorticalSim, which avoids the longstanding inhomogeneity problem stemming from previous, less realistic models for microtubule-based nucleation. We show that microtubule-based nucleation increases the sensitivity of the array to cell geometry, and extends the regime of spontaneous alignment compared to isotropic nucleation. In the case of cylindrical cell shapes, we show that this translates into a strong tendency to align in the transverse direction rather than along the vertical axis, and this is robust against small directional cues favouring the longitudinal direction. Comparing various cylinders and boxes, we show that different nucleation mechanisms result in different preferred array orientations, with the largest differences on cylinders. Our model provides a powerful tool for investigating how plant cells integrate multiple biases to orient their cortical arrays, offering new insights into the biophysical mechanisms underlying cell shape and growth.

biophysics↗

bioRxiv2024

Enhanced extracellular matrix remodeling due to embedded spheroid fluidization

Tumor spheroids are in vitro three-dimensional, cellular collectives consisting of cancerous cells. Embedding these spheroids in an in vitro fibrous environment, such as a collagen network, to mimic the extracellular matrix (ECM) provides an essential platform to quantitatively investigate the biophysical mechanisms leading to tumor invasion of the ECM. To understand the mechanical interplay between tumor spheroids and the ECM, we computationally construct and study a three-dimensional vertex model for a tumor spheroid that is mechanically coupled to a cross-linked network of fibers. In such a vertex model, cells are represented as deformable polyhedrons that share faces. Some fraction of the boundary faces of the tumor spheroid contain linker springs connecting the center of the boundary face to the nearest node in the fiber network. As these linker springs actively contract, the fiber network remodels. By toggling between fluid-like and solid-like spheroids via changing the dimensionless cell shape index, we find that the spheroid rheology affects the remodeling of the fiber network. More precisely, fluid-like spheroids displace the fiber network more on average near the vicinity of the spheroid than solid-like spheroids. We also find more densification of the fiber network near the spheroid for the fluid-like spheroids. These spheroid rheology-dependent effects are the result of cellular motility due to active cellular rearrangements that emerge over time in the fluid-like spheroids to generate spheroid shape fluctuations. These shape fluctuations lead to emergent feedback between the spheroid and the fiber network to further remodel the fiber network with, for example, lower radial alignment of the higher-tensioned fibers given the breaking of spheroidal radial symmetry, which can then further remodel the spheroid. Our results uncover intricate morphological-mechanical interplay between an embedded spheroid and its surrounding fiber network with both spheroid contractile strength and spheroid shape fluctuations playing important roles in the pre-invasion stages of tumor invasion.

biophysics↗

bioRxiv2024

The Drosophila RNA binding protein Hrp48 binds a specificRNA sequence of the msl-2 mRNA 3' UTR to regulatetranslation

Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3 untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3 UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.

biophysics↗

bioRxiv2024

The Hill function is the universal Hopfield barrier for sharpness of input-output responses

The Hill functions, [H]h(x) = xh/(1 + xh), have been widely used in biology for over a century but, with the exception of [H]1, they have had no justification other than as a convenient fit to empirical data. Here, we show that they are the universal limit for the sharpness of any input-output response arising from a Markov process model at thermodynamic equilibrium. Models may represent arbitrary molecular complexity, with multiple ligands, internal states, conformations, co-regulators, etc, under core assumptions that are detailed in the paper. The model output may be any linear combination of steady-state probabilities, with components other than the chosen input ligand held constant. This formulation generalises most of the responses in the literature. We use a coarse-graining method in the graph-theoretic linear framework to show that two sharpness measures for input-output responses fall within an effectively bounded region of the positive quadrant, {Omega}m [sub] ([R]+)2, for any equilibrium model with m input binding sites. {Omega}m exhibits a cusp which approaches, but never exceeds, the sharpness of [H]m but the region and the cusp can be exceeded when models are taken away from thermodynamic equilibrium. Such fundamental thermodynamic limits are called Hopfield barriers and our results provide a biophysical justification for the Hill functions as the universal Hopfield barriers for sharpness. Our results also introduce an object, {Omega}m, whose structure may be of mathematical interest, and suggest the importance of characterising Hopfield barriers for other forms of cellular information processing.

biophysics↗

bioRxiv2024

Elucidating the link between binding statistics and Shannon information in biological cooperative networks

Cooperative response is ubiquitous and vital for regulatory control and ultra-sensitivity in various cellular biophysical processes. Ligands, acting as signaling molecules, carry information which is transmitted through the elements of the biochemical network during binding processes. In this work, we address a fundamental issue regarding the link between the information content of the various states of the binding network and the observable binding statistics. Two seminal models of cooperativity, viz., the Koshland-Nemethy-Filmer (KNF) network and the Monod-Wyman-Changeux (MWC) network are considered for this purpsoe which are solved using the chemical master equation approach. Our results establish that the variation of Shannon information associated with the network states has a generic form related to the average binding number. Further, the logarithmic sensitivity of the slope of Shannon information is shown to be related to the Hill slope in terms of the variance of the binding number distributions. 1

biophysics↗

bioRxiv2024

Phase separation of second prion domain of CPEB3: Insights from the aggregation and structural studies

The maintenance of long-term memory requires sustainable synaptic connections, mediated by the prion-like transformation of the translational regulator protein CPEB3 (Cytoplasmic Polyadenylation Element Binding protein isoform 3) in mammals. The N- terminal prion domain of CPEB3, composed of the two prion subdomains PRD1 and PRD2 has previously been demonstrated to perform a crucial role in imparting prion-like properties to the protein. We have already reported the amyloid-core of the first prion subdomain (PRD1) of the mouse CPEB3. Here, we have investigated the aggregation properties and the structural characteristics of the mouse PRD2 (mPRD2) in vitro. We found that the mPRD2 undergoes phase separation. Interestingly, the mPRD2 formed stable and amyloid-like solid condensates instead of the typical liquid condensate formation. Solid-state NMR and other biophysical studies revealed the existence of mixed secondary structures for mPRD2 in condensates. We propose that the distinct phase separation behaviour of the mPRD2 would be due to the conformational changes attributed to the pattern of the mPRD2 amino acid sequence, resulting in the formation of rigid and amyloid-like self-assembly.

biophysics↗

bioRxiv2024

Selectivity of Complex Coacervation in Multi-Protein Mixtures

Liquid-liquid phase separation of biomolecules is increasingly recognized as relevant to various cellular functions, and complex coacervation of biomacromolecules, particularly proteins, is emerging as a key mechanism for this phenomenon. Complex coacervation is also being explored as a potential protein purification method due to its potential scalability, aqueous operation, and ability to produce a highly concentrated product. However, to date most studies of complex coacervation have evaluated the phase behavior of a binary mixture of two oppositely charged macromolecules. Therefore, a comprehensive understanding of the phase behavior of complex biological mixtures has yet to be established. To address this, a panel of engineered proteins was designed to allow for quantitative analysis of the complex coacervation of individual proteins within a multi-component mixture. The behavior of individual proteins was evaluated using a defined mixture of proteins that mimics the charge profile of the E. coli proteome. To allow for direct quantification of proteins in each phase, spectrally separated fluorescent proteins were used to construct the protein mixture. From this quantitative analysis, we observed that the coacervation behavior of individual proteins in the mixture was consistent with each other, which was distinctive from the behavior when each protein was evaluated in a single-protein system. Subtle differences in biophysical properties between the proteins became noticeable in the mixture, which allowed us to elucidate parameters for protein complex coacervation. With this understanding, we successfully designed methods to enrich a range of proteins of interest from a mixture of proteins.

biophysics↗

bioRxiv2024

Multiple Dynamic Modes of the Bcd Gradient are Explained by Quantum Mechanics

Extracellular diffusion coupled with degradation is considered as the dominant mechanism behind the establishment of morphogen gradients. However, the fundamental nature of these biophysical processes visa viz the Bicoid (Bcd) morphogen gradient remains unclear. Fluorescence correlation spectroscopy (FCS) has recently revealed multiple modes of Bcd transport at different spatial and temporal locations across the embryo. We here show that these observations, and a few others, are fitted by a model fundamentally based on quantum mechanics. We also indicate that the abstract and auxiliary feature called chirality of the said formalism finds a natural expression in our model of the Bcd gradient formation that might be verified in future experiments on the system.

biophysics↗

bioRxiv2024

Compliant DNA Origami Nanoactuators as Size-Selective Nanopores

Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, we harness the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between three stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behaviour with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand - thereby impacting fields as diverse as drug-delivery, biomolecule sorting and sensing, as well as bottom-up synthetic biology.

biophysics↗

bioRxiv2024

Planar Optical Tweezer Trap (2D-LOT) System Realized by Light Sheet Illumination & Orthogonal Widefield Detection

We report the realization of the first planar optical tweezer trap system by a sheet of light. To visualize the trapping of the target object (dielectric bead or live cell) in a plane, an orthogonal widefield detection is employed. The planar / two-dimensional lightsheet optical tweezer (2D-LOT) sub-system is realized in an inverted microscopy mode with illumination from the bottom. A 1064 nm laser (power [~] 500mW) is expanded and directed to a combination of cylindrical lens and high NA objective lens to generate a tightly-focused diffraction-limited light sheet. The object to be trapped is injected in the specimen chamber (consists of two coverslips placed at a distance of {approx} 1 mm) using a syringe. The solution containing the objects stayed in the chamber due to the surface tension of the fluid. The illumination of trap-laser light is along Z-direction (with coverslip along XZ-plane) whereas, the detection is achieved perpendicular to the coverslip (along Y-axis). The orthogonal detection is employed to directly visualize the trapping in a plane. To better visualize the specimen, a separate white light illumination sub-system is used. The characterization of system PSF estimates the size of light sheet trap PSF to be, 2073.84 {micro}m2 which defines the active trap region / area. Beads are tracked on their way to the trap region for determining the trap stiffness along Z and X i.e, kz = 1.13 {+/-} 0.034 pN/{micro}m and kx = 0.74 {+/-} 0.021 pN/{micro}m. Results (image and video) show real-time trapping of dielectric beads in the trap zone (2D plane) generated by the light sheet. The beads can be seen getting trapped from all directions in the XZ-plane. Prolonged exposure to the light sheet builds up a 2D array of beads in the trap zone. Similar experiments on live NIH3T3 cells show cells trapped in the 2D trap. The potential of the planar trap lies in its ability to confine objects in two dimensions, thereby opening new kinds of experiments in biophysics, atomic physics, and optical physics. Statement of SignificanceThe ability to trap and confine objects in two dimensions / a plane is an incredible feat that paves the way for new experiments in physical and biological sciences. Currently, no technique can achieve two-dimensional trapping of objects. Such a trap is realized by a sheet of light generating a near-rectangular potential trap-zone, enabling tweezing confined to a plane. This is unlike existing point-focus-based tweezers that are capable of point-potential well. The technique is expected to have widespread applications in science and engineering.

biophysics↗

bioRxiv2024

Bi-directional allosteric pathway in NMDA receptor activation and modulation

N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors involved in learning and memory. NMDA receptors primarily comprise two GluN1 and two GluN2 subunits. The GluN2 subunit dictates biophysical receptor properties, including the extent of receptor activation and desensitization. GluN2A- and GluN2D-containing receptors represent two functional extremes. To uncover the conformational basis of their functional divergence, we utilized single-molecule fluorescence resonance energy transfer to probe the extracellular domains of these receptor subtypes under resting and ligand-bound conditions. We find that the conformational profile of the GluN2 amino-terminal domain correlates with the disparate functions of GluN2A- and GluN2D-containing receptors. Changes at the pre-transmembrane segments inversely correlate with those observed at the amino-terminal domain, confirming direct allosteric communication between these domains. Additionally, binding of a positive allosteric modulator at the transmembrane domain shifts the conformational profile of the amino-terminal domain towards the active state, revealing a bidirectional allosteric pathway between extracellular and transmembrane domains.

biophysics↗

bioRxiv2024

Detecting directed motion and confinement in single-particle trajectories using hidden variables

Single-particle tracking is a powerful tool for understanding protein dynamics and characterizing microenvironments. As the motion of unconstrained nanoscale particles is governed by Brownian diffusion, deviations from this behavior are biophysically insightful. However, the stochastic nature of particle movement and the presence of localization error pose a challenge for the robust classification of non-Brownian motion. Here, we present aTrack, a versatile tool for classifying track behaviors and extracting key parameters for particles undergoing Brownian, confined, or directed motion. Our tool quickly and accurately estimates motion parameters from individual tracks. Further, our tool can analyze populations of tracks and determine the most likely number of motion states. We show the working range of our approach on simulated tracks and demonstrate its application for characterizing particle motion in cells and for biosensing applications. aTrack is implemented as a stand-alone software, making it simple to analyze track data.

biophysics↗

bioRxiv2024

Morphogenesis of bacterial colonies in polymeric environments

Many bacteria live in polymeric fluids, such as mucus, environmental polysaccharides, and extracellular polymers in biofilms. However, lab studies typically focus on cells in polymer-free fluids. Here, we show that interactions with polymers shape a fundamental feature of bacterial life--how they proliferate in space in multicellular colonies. Using experiments, we find that when polymer is sufficiently concentrated, cells generically and reversibly form large serpentine "cables" as they proliferate. By combining experiments with biophysical theory and simulations, we demonstrate that this distinctive form of colony morphogenesis arises from an interplay between polymer-induced entropic attraction between neighboring cells and their hindered ability to diffusely separate from each other in a viscous polymer solution. Our work thus reveals a pivotal role of polymers in sculpting proliferating bacterial colonies, with implications for how they interact with hosts and with the natural environment, and uncovers quantitative principles governing colony morphogenesis in such complex environments.

biophysics↗

bioRxiv2024

Combined thermodynamic and time-resolved structural analysis of interactions between AP2 and biomimetic plasma membranes provides insights into clathrin-mediated endocytosis

Clathrin mediated endocytosis (CME) is the main mechanism for swift and selective uptake of proteins into eukaryotic cells. CME is initiated by recruitment to the plasma membrane (PM) of the adaptor protein AP2, which recognizes the PM-associated lipid PtdIns(4,5)P2, as well as the protein cargo to be internalized. Nonetheless, many aspects of this process remain unclear due to their in vivo complexity. Here, a thermodynamic and time-resolved structural analysis of AP2 binding to different biomimetic PM was undertaken under physiological conditions using a combination of neutron reflectometry, interfacial tensiometry and rheology, and atomic force microscopy. The resultant in vitro data replicated previous in vivo observations, as well as yielded biophysical insights into normal and aborted CME. The presence of cargo may not be pivotal for the "activating" conformational change of AP2. However, the presence of cargo extends AP2s residence time on the membrane surface, due to slower on- and off-rates, thereby tentatively giving sufficient time for CME to proceed fully. Moreover, upon interaction with AP2, phospholipid lateral diffusion decreases markedly, inducing a gel phase attributed to creating a percolated network involving AP2 on the membrane, which could potentially serve as a mechanism for modulating subsequent clathrin binding.

biophysics↗

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