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Results for “biophysics”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2021

Understanding the interaction of 14-3-3 proteins with hDMX and hDM2: a structural and biophysical study

p53 plays a critical role in regulating diverse biological processes: DNA repair, cell cycle arrest, apoptosis, and senescence. The p53 pathway has therefore served as the focus for drug-discovery efforts. p53 is negatively regulated by hDMX and hDM2; prior studies have identified 14-3-3 proteins as hDMX and hDM2 client proteins. 14-3-3 proteins are adaptor proteins that modulate localisation, degradation and interactions of their targets in response to phosphorylation. Thus 14-3-3 proteins may indirectly modulate the interaction between hDMX or hDM2 and p53 and represent potential targets for modulation of the p53 pathway. In this manuscript we report on the biophysical and structural characterization of peptide/protein interactions that are representative of the interaction between 14-3-3 and hDMX or hDM2. The data establish that proximal phosphosites spaced [~]20-25 residues apart in both hDMX and hDM2 co-operate to facilitate high-affinity 14-3-3 binding and provide structural insight that can be utilized in future stabilizer/inhibitor discovery efforts.

biophysics↗

bioRxiv2022

GNNQQNY: Methodology for biophysical and structural understanding of aggregation

GNNQQNY sequence offers crucial information about the formation and structure of an amyloid fibril. In this study, we demonstrate a reproducible solubilisation protocol where the reduction of pH to 2.0 resulted in the generation of GNNQQNY monomers. The subsequent ultracentrifugation step removes the residual insoluble peptide from the homogeneous solution. This procedure ensures and allows the peptides to remain monomers till their aggregation is triggered by adjusting the pH to 7.2. The aggregation kinetics analysis showed a distinct lag-phase that is concentration-dependent, indicating nucleation-dependent aggregation kinetics. Nucleation kinetics analysis suggested a critical nucleus of size [~]7 monomers at physiological conditions. The formed nucleus acts as a template for further self-assembly leading to the formation of highly ordered amyloid fibrils. These findings suggest that the proposed solubilisation protocol provides the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation in GNNQQNY sequences. This procedure can also be used for solubilising such small amyloidogenic sequences for their biophysical studies.

biophysics↗

bioRxiv2022

Buforin II-Escherichia coli's DNA interactome: Detailed biophysical characterization revealed nanoscale complexes likely formed by DNA supercoiling

Antimicrobial peptides (AMPs) have emerged as exciting alternatives to the alarming increase of multiresistant bacteria due to their high activity against them through mechanisms that are thought to largely avoid resistance in the long term. Buforin II (BUFII) is an antibacterial peptide hypothesized to kill bacteria by crossing their membranes to interact with intracellular molecules and interrupt key processes for survival. In particular, interactions with DNA have been considered crucial for triggering cell death mechanisms. However, such interactions are still unknown, and thus far, no reports are available describing BUFII-DNA complexes. Here, we describe a complete biophysical study of the interaction between BUFII and Escherichia coli gDNA via spectrofluorimetric, spectroscopic, and microscopic techniques, complemented with whole-genome sequencing. The E. colis DNA-BUFII interactome was isolated by an in vitro pull-down method aided by BUFII-magnetite nanobioconjugates. Our results demonstrated that DNA-BUFII formed round-shape nanoscale complexes by strong electrostatic interactions, likely occurring nonspecifically throughout the entire bacterial genome. Further sequencing of the isolated DNA fragments corroborated this notion and led to hypothesize that BUFII is possibly responsible for inducing DNAs supercoiling. Other evidence for this idea was provided by the significant DNA conformational changes observed upon interaction with BUFII. Even though the evidence found fails to describe the complete action mechanism of BUFII in vivo, our findings pave the way to engineer DNA-peptide supramolecular complexes very precisely, which might find application in the field of gene therapy delivery.

biophysics↗

bioRxiv2022

A biophysical, minimal model to investigate age-related changes in CA1 pyramidal cell electrical activity

Aging is a physiological process that is still poorly understood, especially with respect to effects on the brain. There are open questions about aging that are difficult to answer with an experimental approach. Underlying challenges include the difficulty of recording in vivo single cell and network activity simultaneously with submillisecond resolution, and brain compensatory mechanisms triggered by genetic, pharmacologic, or behavioral manipulations. Mathematical modeling can help address some of these questions by allowing us to fix parameters that cannot be controlled experimentally and investigate neural activity under different conditions. We present a biophysical minimal model of CA1 pyramidal cells (PCs) based on general expressions for transmembrane ion transport derived from basic thermodynamical principles. The model allows directly varying the contribution of ion channels by changing their number. By analyzing the dynamics of the model, we find parameter ranges that reproduce the variability in electrical activity seen in PCs. In addition, increasing the L-type Ca2+ channel expression in the model reproduces age-related changes in excitability that are qualitatively and quantitatively similar to those observed in PCs from aged animals.

biophysics↗

bioRxiv2022

Biophysical and Structural Characterization of a Viral Genome Packaging Motor

Like many dsDNA viruses, bacteriophage {lambda} replicates its genome as a concatemer consisting of multiple copies of covalently linked dsDNA genomes. To encapsidate a single genome within a nascent procapsid, {lambda} must: 1) find its own dsDNA amongst the multitude of host nucleic acids; 2) identify the genomic start site; 3) cut the DNA; 4) bring the excised DNA to a procapsid; 5) translocate DNA into the capsid; 6) cut DNA again at a packaging termination site, 7) disengage from the newly filled capsid; and 8) bring the remainder of the genomic concatemer to fill another empty procapsid. These disparate genome processing tasks are carried out by a single virus-encoded enzyme complex called terminase. While it has been shown that {lambda} terminase initially forms a tetrameric complex to cut DNA, it is not clear whether the same configuration translocates DNA. Here, we describe biophysical and initial structural characterization of a {lambda} terminase translocation complex. Analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS) indicate that between 4 and 5 protomeric subunits assemble a cone-shaped terminase complex with a maximum dimension of [~]230 and radius of gyration of [~]72 [A]. Two-dimensional classification of cryoEM images of {lambda} terminase are consistent with these dimensions and show that particles assume a preferred orientation in ice. The orientations appear to be end-on, as terminase rings resemble a starfish with approximate pentameric symmetry. While [~]5-fold symmetry is apparent, one of the five "arms" appears partially displaced with weaker more diffuse density in some classes, suggesting flexibility and/or partial occupancy. Charge detection mass spectrometry (CDMS) is consistent with a pentameric complex, with evidence that one motor subunit is weakly bound. Kinetic analysis indicates that the complex hydrolyzes ATP at a rate comparable to the rates of other phage packaging motors. Together with previously published data, these results suggest that {lambda} terminase assembles conformationally and stoichiometrically distinct complexes to carry out different genome processing tasks. We propose a "symmetry resolution" pathway to explain how terminase transitions between these structurally and functionally distinct states.

biophysics↗

bioRxiv2022

Biophysical modeling of actin-mediated structural plasticity reveals mechanical adaptation in dendritic spines

Synaptic plasticity is important for learning and memory formation; it describes the strengthening or weakening of connections between synapses. The postsynaptic part of excitatory synapses resides in dendritic spines, which are small protrusions on the dendrites. One of the key features of synaptic plasticity is its correlation with the size of these spines. A long-lasting synaptic strength increase (long-term potentiation, LTP) is only possible through the reconfiguration of the actin spine cytoskeleton. Here, we develop an experimentally-informed three-dimensional computational model in a moving boundary framework to investigate this reconfiguration. Our model describes the reactions between actin and actin-binding proteins (ABPs) leading to the cytoskeleton remodeling and their effect on the spine membrane shape to examine the spine enlargement upon LTP. Moreover, we find that the incorporation of perisynaptic elements enhances spine enlargement upon LTP, exhibiting the importance of accounting for these elements when studying structural LTP. Our model shows adaptation to repeated stimuli resulting from the interactions between spine proteins and mechanical forces. Significance StatementDendritic spines are small protrusions that receive stimulation from presynaptic neurons. Upon stimulation, the dendritic spines change their size, an important feature of synaptic plasticity. This change is achieved by modifications to the actin cytoskeleton and mediated by many actin-binding proteins. To investigate the fundamental mechanics of spine expansion, we developed a 3D biophysical model that accounts for the dynamics of cytoskeleton-membrane interactions. Our simulations predict that spine expansion due to actin remodeling can be enhanced by including the interaction with perisynaptic elements that affect the spines mechanical properties. We also found that mechanical properties can control spine expansion after repeated stimuli, which ensures physiological size. Thus, we predict that spine growth is regulated by its mechanical properties.

biophysics↗

bioRxiv2022

Genome-wide analysis of the dynamic and biophysical properties of chromatin and nuclear proteins in living cells with Hi-D

To understand the dynamic nature of the genome in real-time, the localization and rearrangement of DNA and DNA-binding proteins must be analyzed across the entire nucleus of single living cells. Recently, we developed a new computational light microscopy technique, called high-resolution diffusion mapping (Hi-D), that can accurately detect, classify, and map the types of diffusion and biophysical parameters such as the diffusion constant, anomalous exponent, drift velocity, and physical diffusion models at a high spatial resolution over the entire genome in living cells. Hi-D combines dense optical flow to detect and track local chromatin and protein motion, and Bayesian inference to characterize this local movement at nanoscale resolution. The initial implementation requires solid experience using MATLAB (MathWorks) and computational resources, for instance, access to a computer cluster, to perform the Hi-D analysis. In addition, this implementation takes [~]18-24 hours to analyze a typical imaging stack. To avoid these limitations and emphasize high-performance implementation, we present a customized version called Hi-D-Py. The new implementation is written in the open-source Python programming language and has an option for parallelizing the calculations to run on multi-core CPUs. The functionality of Hi-D-Py is exposed to the users via user-friendly documented Python notebooks. Our efficient implementation reduces the analysis time to less than one hour using a multi-core CPU with a single compute node. We also present different applications of Hi-D for live-imaging of DNA, H2B, and RNA Pol II sequences acquired with spinning disk confocal and super-resolution structured illumination microscopy.

biophysics↗

bioRxiv2022

Tissue Forge: Interactive Biological and Biophysics Simulation Environment

Tissue Forge is an open-source interactive environment for particle-based physics, chemistry and biology modeling and simulation. Tissue Forge allows users to create, simulate and explore models and virtual experiments based on soft condensed matter physics at multiple scales, from the molecular to the multicellular, using a simple, consistent interface. While Tissue Forge is designed to simplify solving problems in complex subcellular, cellular and tissue biophysics, it supports applications ranging from classic molecular dynamics to agent-based multicellular systems with dynamic populations. Tissue Forge users can build and interact with models and simulations in real-time and change simulation details during execution, or execute simulations off-screen and/or remotely in high-performance computing environments. Tissue Forge provides a growing library of built-in model components along with support for user-specified models during the development and application of custom, agent-based models. Tissue Forge includes an extensive Python API for model and simulation specification via Python scripts, an IPython console and a Jupyter Notebook, as well as C and C++ APIs for integrated applications with other software tools. Tissue Forge supports installations on 64-bit Windows, Linux and MacOS systems and is available for local installation via conda. 1 Author SummaryTissue Forge is a physics-based modeling and simulation software environment for research problems in physics, chemistry and biology. Tissue Forge supports modeling at a wide range of scales, from as small as the sub-nanometer, to as large as hundreds of micrometers, using particle-based models. It provides rich features for simulation development and application at all stages of model-based research, like real-time simulation visualization and interactivity, and off-screen batch execution, rendering, and GPU acceleration. Users can employ built-in models to represent a wide variety of physical processes, like chemical reactions, fluid convection and intercellular adhesion, or define their own models for agentand rule-based modeling. Tissue Forge is open-source, free and easy to install, supports simulation development in C, C++ and Python programming languages, and can be used as integrated software or in an interactive IPython console and Jupyter Notebook. Tissue Forge also provides a dedicated space for application-specific and user-contributed modeling and simulation features, and developers are welcome to contribute their custom features for distribution in future releases.

biophysics↗

bioRxiv2022

Structure adaptation in Omicron SARS-CoV-2/hACE2:Biophysical origins of evolutionary driving forces

Since its emergence, the Covid19 pandemic has been sustained by a series of transmission waves initiated by new variants of the SARS-CoV-2 virus. Some of these arise with higher transmissivity and/or increased disease severity. Here we use molecular dynamics simulations to examine the modulation of the fundamental interactions between the receptor binding domain (RBD) of the spike glycoprotein and the host cell receptor (human angiotensin-converting enzyme 2: hACE2) arising from Omicron variant mutations (BA.1 and BA.2) relative to the original wild type strain. We find significant structural differences in the complexes which overall bring the spike protein and its receptor into closer proximity. These are consistent with and attributed to the higher positive charge on the RBD conferred by BA.1 and BA.2 mutations relative to the wild type. However, further differences between sub-variants BA.1 and BA.2 (which have equivalent RBD charges) are also evident: Mutations affect interdomain interactions between the up-chain and its clockwise neighbor chain, resulting in enhanced flexibility for BA.2. Consequently, additional close contacts arise in BA.2 which include binding to hACE2 by a second spike protein monomer, in addition to the up-chain - a motif not found in BA.1. Finally, the mechanism by which the glycans stabilize the up state of the Spike protein differs for the wild type and the Omicrons. We also found the glycan on N90 of hACE2 turns from inhibiting, to facilitating the binding to Omicron spike protein. These structural and electrostatic differences offer further insight into the mechanisms by which viral mutations modulate host cell binding and provide a biophysical basis for evolutionary driving forces.

biophysics↗

bioRxiv2023

The full spectrum of OCT1 (SLC22A1) mutations bridges transporter biophysics to drug pharmacogenomics

Membrane transporters play a fundamental role in the tissue distribution of endogenous compounds and xenobiotics and are major determinants of efficacy and side effects profiles. Polymorphisms within these drug transporters result in inter-individual variation in drug response, with some patients not responding to the recommended dosage of drug whereas others experience catastrophic side effects. For example, variants within the major hepatic Human organic cation transporter OCT1 (SLC22A1) can change endogenous organic cations and many prescription drug levels. To understand how variants mechanistically impact drug uptake, we systematically study how all known and possible single missense and single amino acid deletion variants impact expression and substrate uptake of OCT1. We find that human variants primarily disrupt function via folding rather than substrate uptake. Our study revealed that the major determinants of folding reside in the first 300 amino acids, including the first 6 transmembrane domains and the extracellular domain (ECD) with a stabilizing and highly conserved stabilizing helical motif making key interactions between the ECD and transmembrane domains. Using the functional data combined with computational approaches, we determine and validate a structure-function model of OCT1s conformational ensemble without experimental structures. Using this model and molecular dynamic simulations of key mutants, we determine biophysical mechanisms for how specific human variants alter transport phenotypes. We identify differences in frequencies of reduced function alleles across populations with East Asians vs European populations having the lowest and highest frequency of reduced function variants, respectively. Mining human population databases reveals that reduced function alleles of OCT1 identified in this study associate significantly with high LDL cholesterol levels. Our general approach broadly applied could transform the landscape of precision medicine by producing a mechanistic basis for understanding the effects of human mutations on disease and drug response.

biophysics↗

bioRxiv2023

The biophysical basis of bacterial colony growth

Bacteria often attach to surfaces and grow densely-packed communities called biofilms. As biofilms grow, they expand across the surface, increasing their surface area and access to nutrients. Thus, the overall growth rate of a biofilm is directly dependent on its "range expansion" rate. One factor that limits the range expansion rate is vertical growth; at the biofilm edge there is a direct trade-off between horizontal and vertical growth--the more a biofilm grows up, the less it can grow out. Thus, the balance of horizontal and vertical growth impacts the range expansion rate and, crucially, the overall biofilm growth rate. However, the biophysical connection between horizontal and vertical growth remains poorly understood, due in large part to difficulty in resolving biofilm shape with sufficient spatial and temporal resolution from small length scales to macroscopic sizes. Here, we experimentally show that the horizontal expansion rate of bacterial colonies is controlled by the contact angle at the biofilm edge. Using white light interferometry, we measure the three-dimensional surface morphology of growing colonies, and find that small colonies are surprisingly well-described as spherical caps. At later times, nutrient diffusion and uptake prevent the tall colony center from growing exponentially. However, the colony edge always has a region short enough to grow exponentially; the size and shape of this region, characterized by its contact angle, along with cellular doubling time, determines the range expansion rate. We found that the geometry of the exponentially growing biofilm edge is well-described as a spherical-cap-napkin-ring, i.e., a spherical cap with a cylindrical hole in its center (where the biofilm is too tall to grow exponentially). We derive an exact expression for the spherical-cap-napkin-ring-based range expansion rate; further, to first order, the expansion rate only depends on the colony contact angle, the thickness of the exponentially growing region, and the cellular doubling time. We experimentally validate both of these expressions. In line with our theoretical predictions, we find that biofilms with long cellular doubling times and small contact angles do in fact grow faster than biofilms with short cellular doubling times and large contact angles. Accordingly, sensitivity analysis shows that biofilm growth rates are more sensitive to their contact angles than to their cellular growth rates. Thus, to understand the fitness of a growing biofilm, one must account for its shape, not just its cellular doubling time.

biophysics↗

bioRxiv2024

Biophysical modeling of membrane curvature generation and curvature sensing by the glycocalyx

Generation of membrane curvature is fundamental to cellular function. Recent studies have established that the glycocalyx, a sugar-rich polymer layer at the cell surface, can generate membrane curvature. While there have been some theoretical efforts to understand the interplay between the glycocalyx and membrane bending, there remain open questions about how the properties of the glycocalyx affect membrane bending. For example, the relationship between membrane curvature and the density of glycosylated proteins on its surface remains unclear. In this work, we use polymer brush theory to develop a detailed biophysical model of the energetic interactions of the glycocalyx with the membrane. Using this model, we identify the conditions under which the glycocalyx can both generate and sense curvature. Our model predicts that the extent of membrane curvature generated depends on the grafting density of the glycocalyx and the length of the polymers constituting the glycocalyx. Furthermore, when coupled with the intrinsic membrane properties such as spontaneous curvature and a line tension along the membrane, the curvature generation properties of the glycocalyx are enhanced. These predictions were tested experimentally by examining the propensity of glycosylated transmembrane proteins to drive the assembly of highly-curved filopodial protrusions at the plasma membrane of adherent mammalian cells. Our model also predicts that the glycocalyx has curvature sensing capabilities, in agreement with the results of our experiments. Thus, our study develops a quantitative framework for mapping the properties of the glycocalyx to the curvature generation capability of the membrane. Significance StatementThe glycocalyx is a dense layer of glycosylated transmembrane proteins and lipids distributed on the extracellular surface of eukaryotic cells. It is known to mediate cell-cell interactions and protect cells from invasion by pathogens. However, recently it has been found to play a role in generating membrane curvature, which is essential to diverse cellular functions spanning from endocytosis to cell division. Experiments have revealed that different membrane shapes: spherical vesicles, pearls, and tubes are regulated by the glycocalyx. However, currently, we lack a quantitative physical explanation of how glycocalyx properties determine membrane geometries. Here we develop a polymer brush theory-based model, which suggests that the interplay between glycocalyx polymers and membrane bending captures the wide variety of membrane shapes from spherical buds to elongated pearl-like shapes found in previously published experiments. We predict that the physical properties of glycocalyx polymers, line tension, and membrane elastic parameters play significant roles in regulating membrane morphologies. We find that the glycocalyx prefers higher local membrane curvature indicating that the glycocalyx can sense local curvature.

biophysics↗

bioRxiv2024

LLPS REDIFINE allows the biophysical characterization of multicomponent condensates without tags or labels

Liquid-liquid phase separation (LLPS) phenomenon plays a vital role in multiple cell biology processes, providing a mechanism to concentrate biomolecules and promote cellular reactions locally. Despite its significance in biology, there is a lack of conventional techniques suitable for studying biphasic samples in their biologically relevant form. Here, we present a label-free and non-invasive approach to characterize protein, RNA and water in biomolecular condensates termed LLPS REstricted DIFusion of INvisible speciEs (REDIFINE). Relying on diffusion NMR measurements, REDIFINE exploits the exchange dynamics between the condensed and dispersed phases to allow the determination of not only diffusion constants in both phases but also the fractions of the species, the average radius of the condensed droplets and the exchange rate between the phases. We can also access the concentration of proteins in both phases. Observing proteins, RNAs, water, and even small molecules, REDIFINE analysis allows a rapid biophysical characterization of multicomponent condensates which is important to understand their functional roles. In comparing multiple systems, REDIFINE reveals that folded RNA-binding proteins form smaller and more dynamic droplets compared to the disordered ones. In addition, REDIFINE proved to be valuable beyond LLPS for the determination of binding constants in soluble protein-RNA without the need for titration.

biophysics↗

bioRxiv2024

In silico biophysics and rheology of blood and red blood cells in Gaucher Disease

Gaucher Disease (GD) is a rare genetic disorder characterized by a deficiency in the enzyme glucocerebrosidase, leading to the accumulation of glucosylceramide in various cells, including red blood cells (RBCs). This accumulation results in altered biomechanical properties and rheological behavior of RBCs, which may play an important role in blood rheology and the development of bone infarcts, avascular necrosis (AVN) and other bone diseases associated with GD. In this study, dissipative particle dynamics (DPD) simulations are employed to investigate the biomechanics and rheology of blood and RBCs in GD under various flow conditions. The model incorporates the unique characteristics of GD RBCs, such as decreased deformability and increased aggregation properties, and aims to capture the resulting changes in RBC biophysics and blood viscosity. This study is the first to explore the Youngs modulus and aggregation parameters of GD RBCs by validating simulations with confocal imaging and experimental RBC disaggregation thresholds. Through in silico simulations, we examine the impact of hematocrit, RBC disaggregation threshold, and cell stiffness on blood viscosity in GD. The results reveal three distinct domains of GD blood viscosity based on shear rate: the aggregation domain, where the RBC disaggregation threshold predominantly influences blood viscosity; the transition area, where both RBC aggregation and stiffness impact on blood viscosity; and the stiffness domain, where the stiffness of RBCs emerges as the primary determinant of blood viscosity. By quantitatively assessing RBC deformability, RBC disaggregation threshold, and blood viscosity in relation to bone disease, we find that the RBC aggregation properties, as well as their deformability and blood viscosity, may contribute to its onset. These findings enhance our understanding of how changes in RBC properties impact on blood viscosity and may affect bone health, offering a partial explanation for the bone complications observed in GD patients. Author summaryIn Gaucher Disease (GD), a genetic deficiency in the enzyme glucocerebrosidase leads to the accumulation of glucosylceramide in red blood cells (RBCs), resulting in altered biomechanical properties. These changes affect blood flow characteristics, particularly blood viscosity, and may contribute to bone health issues seen in GD patients, including bone infarcts, avascular necrosis (AVN), and other bone diseases. In our study, we apply dissipative particle dynamics (DPD) simulations to explore how GD impacts RBC behavior under various flow conditions. We model GD RBCs with decreased deformability and increased aggregation, examining how these properties influence blood viscosity across three distinct shear rate domains: aggregation, transition, and stiffness. By validating our simulations with confocal imaging data and experimental RBC disaggregation thresholds, we quantitatively assess the effects of RBC stiffness, aggregation, and hematocrit levels on blood flow in GD. We find that the RBC aggregation properties, deformability and blood viscosity, may contribute to the onset of bone disease. These findings improve our understanding of how changes in RBC properties influence blood viscosity and may contribute to bone health issues, providing a partial explanation for the bone complications observed in GD patients.

biophysics↗

bioRxiv2024

Cell surface crowding is a tunable biophysical barrier to cell-cell fusion

Cell-cell fusion is fundamental to developmental processes such as muscle formation, as well as to viral infections that cause pathological syncytia. An essential step in fusion is close membrane apposition, but cell membranes are crowded with proteins, glycoproteins, and glycolipids, all of which must be cleared before a fusion pore can be nucleated. Here, we find that cell surface crowding drastically reduces fusogenicity in multiple systems, independent of the method for driving fusion. We estimate that cell surface crowding presents an energetic barrier to membrane apposition on the scale of [~]100kBT, greater than that of bare membrane fusion. We show that increasing cell surface crowding reduces fusion efficiency of PEG-mediated and fusogen-mediated cell-cell fusion, as well as synthetic membranes under force. Interestingly, we find that differentiating myoblasts naturally decrease cell surface crowding prior to fusion. Cell surface crowding presents an underappreciated biophysical barrier that may be tuned developmentally and could be targeted externally to control tissue-specific cell-cell fusion.

biophysics↗

bioRxiv2025

Biophysical Basis of the in vivo Electroretinogram of the Mouse: 4-Shell Current Source Density Model

A goal of contemporary physiology is to translate the knowledge obtained ex vivo of the molecular structure and function of ionic mechanisms into tools for quantitative, in vivo measurement of that function in humans, and in animal models of disease and therapeutic intervention. Non-invasive field potentials such as ECGs, EMGs, EEGs, and ERGs hold great promise in efforts to achieve this goal, but their full translation is challenging due to the multiplicity of cells with distinct ionic mechanisms and distributions of membrane current sources and sinks, and the requirement of adequate characterization of volume conduction in the relevant tissue(s). The molecular identities and subcellular distributions of the ionic mechanisms of mouse rod photoreceptors and the adjacent retinal pigment epithelium (RPE) have been thoroughly characterized, and are associated with two major components of the ERG, the a-wave and the c-wave, respectively. To develop a molecular-biophysical description of these components of the ERG, we have pharmacologically and genetically isolated rod photoreceptor-driven currents, created a 3D "4-shell" model of volume conduction in the mouse eye and extraocular tissue, and solved the Equation of Continuity for trans-photoreceptor layer and trans-RPE layer sources. Corneal and intraocular measurements of a- and c-waves are shown to reject the classic Rodieck-Ford electrical circuit model of the ERG, but found consistent with a 4-shell model having realistic values for extracellular conductivity in the eye and extraocular tissues. Our results and analysis explain the large variation across studies in maximal a-wave amplitudes and show how a-wave amplitudes exceeding 1 mV can be achieved.

biophysics↗

bioRxiv2025

Biophysical Basis of the in vivo Electroretinogram of the Mouse: Current Source Density Analysis of Genetically and Pharmacologically Isolated Rod photoreceptor-driven Currents

To better understand the molecular basis of the mouse electroretinogram (ERG) we have developed a biophysical model of the rod photoreceptor layers ionic mechanisms and applied current source-density (CSD) analysis to predict the genetically and pharmacologically isolated rod ERG a-wave. The saturating a-wave is characterized by a rapid relaxation ({tau} ~ 6 ms) from its maximum that has been hypothesized to be caused by HCN1 channel opening consequent to light-triggered sustained hyperpolarization, or by extracellular flow of capacitive current during hyperpolarization. To test these hypotheses, the CSD model included an ensemble of 11 rods with cell body locations spanning the outer nuclear layer, and with ionic mechanisms -- including CNG channels, NCKX in the outer segment, NKX, Kv2.1 channels in the inner segment, and HCN1 throughout the "non-outer segment" -- fully specified as to axial distributions, voltage dependencies, and the extracellular conductivity of the photoreceptor layer extracellular space. Predicting with CSD the steady-state axial distributions of rod dark current and transretinal potential, and the spatio-temporal response of the activation phase of the rods light response to intense stimuli, the analysis confirmed the Robson-Frishman hypothesis that extracellular capacitive current flowing towards the inner segment upon the rapid closure of CNG current plays a major role in shaping the early a-wave. The CSD analysis also reveals that extracellular current from HCN1 channels opened by the hyperpolarization contributes a net extracellular current flowing toward the ONL that contributes materially to the saturated a-wave relaxation and to setting its plateau level.

biophysics↗

bioRxiv2025

An image-based biophysical model of the lung to investigate the effect of pulmonary surfactant on lung function

Lung biomechanics aims to understand the structure-function relationship in the lung under normal and pathological conditions. Pulmonary surfactant has a key structural role in lung function with a significant contribution to the mechanical response of the lungs during respiration. Pulmonary surfactant dynamically regulates surface tension at the air-liquid interface to decrease stiffness during respiration and prevent alveolar collapse during lower volumes. Many lung injuries involve alterations to the contribution of surfactants to lung function. We developed a novel biophysical model using a poroelastic formulation that incorporates pulmonary surfactant dynamics and aims to quantify the contribution of the pulmonary surfactant toward lung compliance. The effect of pulmonary surfactant was modeled as a surface energy function, and the surface behavior was converted to bulk behavior by assuming uniform spherical alveoli. The model was used to simulate respiration and investigate the effect of altered surface tension caused by surfactant dysfunction. The model captured the characteristic sigmoidal inspiratory pressure-volume curve and hysteresis observed during clinical measurements. In addition, the model predicted the expected behavior in surfactant dysfunction in lung injuries. We expect this work to serve as an essential step towards de-convoluting and predicting the contributions of the lung parenchyma and pulmonary surfactant to global and regional lung compliance in health and disease.

biophysics↗