Determining sex-driven excitation-contraction coupling mechanisms in young populations

Biophysical Journal

Intracellular fibroblast growth factors (iFGFs) are key regulators of voltage-gated sodium channels. In heart failure (HF), the late sodium current (I NaL) is markedly enhanced and contributes to arrhythmogenic action potentials (APs). However, I NaL regulation by iFGFs and its therapeutic targeting in HF have not been previously studied. We aimed to investigate the expression of iFGF11-14 splice isoforms and tested for the effects of an iFGF12 A-mimicking inhibitory peptide (FixR) on I NaL magnitude and AP duration in rabbit and murine HF ventricular myocytes. We found that the expression of long iFGF isoforms (eg, iFGF12 A and iFGF13 S), which can inhibit I NaL, was markedly reduced both in a pressure/volume-overload induced HF rabbit model and in a transverse aortic constriction (TAC) induced murine HF model. I NaL density was significantly increased in HF myocytes, and importantly, cell pretreatment …

bioRxiv

Background and AimsSubstantial sex-based differences have been reported in atrial fibrillation (AF), with female patients experiencing worse symptoms, increased complications from drug side effects or ablation, and elevated risk of AF-related stroke and mortality. Recent studies revealed sex-specific alterations in AF-associated Ca2+ dysregulation, whereby female cardiomyocytes more frequently exhibit potentially proarrhythmic Ca2+-driven instabilities compared to male cardiomyocytes. In this study, we aim to gain a mechanistic understanding of the Ca2+-handling disturbances and Ca2+-driven arrhythmogenic events in males vs females and establish their responses to Ca2+-targeted interventions.Methods and ResultsWe incorporated known sex differences and AF-associated changes in the expression and phosphorylation of key Ca2+-handling proteins and in ultrastructural properties and dimensions of atrial cardiomyocytes into our recently developed 3D atrial cardiomyocyte model that couples electrophysiology with spatially detailed Ca2+-handling processes. Our simulations of quiescent cardiomyocytes show increased incidence of Ca2+ sparks in female vs male myocytes in AF, in agreement with previous experimental reports. Additionally, our female model exhibited elevated propensity to develop pacing-induced spontaneous Ca2+ releases (SCRs) and augmented beat-to-beat variability in action potential (AP)-elicited Ca2+ transients compared with the male model. Parameter sensitivity analysis uncovered precise arrhythmogenic contributions of each component that was implicated in sex and/or AF alterations. Specifically …

Cardiovascular Research

Aim Sex-differences in heart failure with preserved ejection fraction (HFpEF) are important, but key mechanisms involved are incompletely understood. While animal models can inform about sex-dependent cellular and molecular changes, many previous preclinical HFpEF models have failed to recapitulate sex-dependent characteristics of human HFpEF. We tested for sex-differences in HFpEF using a two-hit mouse model (leptin receptor-deficient db/db mice plus aldosterone infusion for 4 weeks; db/db+Aldo). Methods and Results We performed echocardiography, electrophysiology, intracellular Ca2+ imaging, and protein analysis. Female HFpEF mice exhibited more severe diastolic dysfunction in line with increased titin N2B isoform expression and PEVK element phosphorylation, and reduced troponin-I phosphorylation. Female HFpEF mice had lower BNP levels than …

Biophysical Journal

The cardiac voltage-gated sodium channel, Na V 1.5, is responsible for action potential initiation and propagation within the heart. Deficits in Na V 1.5 inactivation result in an enhanced, sustained Na+ current (late Na+ current), which is an important contributor to multiple inherited and acquired arrhythmias. Considerable effort has been directed toward the development of selective late Na+ current inhibitors; however, this endeavor has proven to be challenging. Here, we undertake a targeted approach by combined insights from the biophysical mechanism of inactivation with computational protein design to generate a de novo peptide inhibitor of late Na+ current. We show that our designed peptide binds to the desired site, the Na V 1.5 C-terminal domain (CTD), with a submicromolar affinity. Electrophysiology recordings reveal that it effectively inhibits pathogenic late Na+ current arising from either disease-linked …

Biophysical Journal

Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome that drastically reduces patients' quality of life and increases the propensity for lethal ventricular arrhythmias. Importantly, patients' survival rate after first hospitalization is very limited, as most pharmacological options for HFpEF are currently inadequate. Functional changes of ion channels and transporters within the cardiomyocyte can induce both contractile dysfunction and arrhythmias, but exactly how excitation-contraction coupling is altered in HFpEF is still unclear. To address this gap, we implemented an integrative computational/experimental approach aiming at identifying the key disease-associated alterations in ventricular myocytes isolated from our two-hit mouse model of HFpEF (ie, leptin receptor-deficient db/db mice+ aldosterone infusion, db/db+ Aldo). We developed a preliminary parameterization of the mathematical model of …

bioRxiv

Background Heart rhythm relies on complex interactions between the electrogenic membrane proteins and intracellular Ca2+ signaling in sinoatrial node (SAN) myocytes; however, the mechanisms underlying the functional organization of the proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, however the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND). Methods In vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca2+ imaging, immunofluorescent and electron microscopy analysis were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking. Results In both mouse and human SANs, caveolae compartmentalized HCN4, Cav1.2, Cav1.3, Cav3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 …

Biophysical Journal

386a Tuesday, February 13, 2024 expressed in the LM and ID, with low expression at the t-tubules. A similar pattern is observed for iFGF13, the predominant iFGF in the murine heart, and iFGF14. iFGF11 was not detected in murine myocytes, while iFGF12s were found throughout the cells but at a much lower expression. The expression of NaV1. 1 was evenly distributed to the t-tubules, while NaV1. 6 and NaV1. 8 were concentrated in the t-tubules close to the LM. These results suggest various subpopulations of NaV channel and iFGF complexes within each subcellular microdomain. In the future, we will study how different NaV channels and iFGFs colocalize and whether their interactions are altered in diseased cardiomyocytes.

Biophysical Journal

Atrial fibrillation (AF) is the most common arrhythmia worldwide. Though AF affects men and women at a similar rate there are major differences in epidemiology, manifestation, pathophysiology, treatment, and outcomes between sexes. Notably, men are more likely to be diagnosed with AF at an earlier age, and women are more likely to have worse AF symptoms that typically last longer and are more severe. Overall, this leads to a poorer quality of life and worse prognosis for women with AF compared to men. Studies investigating sex-related differences in the pathophysiology underlying AF have so far been limited, and thus the sex-specific mechanisms remain poorly understood. To address these knowledge gaps, we developed biophysically detailed mechanistic models of cardiac myocyte and tissue electrophysiology that integrate current knowledge of ion channel profiles and calcium (Ca) handling properties …

Scientific Reports

Mitochondrial Ca2+ overload can mediate mitochondria-dependent cell death, a major contributor to several human diseases. Indeed, Duchenne muscular dystrophy (MD) is driven by dysfunctional Ca2+ influx across the sarcolemma that causes mitochondrial Ca2+ overload, organelle rupture, and muscle necrosis. The mitochondrial Ca2+ uniporter (MCU) complex is the primary characterized mechanism for acute mitochondrial Ca2+ uptake. One strategy for preventing mitochondrial Ca2+ overload is deletion of the Mcu gene, the pore forming subunit of the MCU-complex. Conversely, enhanced MCU-complex Ca2+ uptake is achieved by deleting the inhibitory Mcub gene. Here we show that myofiber-specific Mcu deletion was not protective in a mouse model of Duchenne MD. Specifically, Mcu gene deletion did not reduce muscle histopathology, did not improve muscle function, and did not prevent mitochondrial …

Biophysical Journal

Intracellular fibroblast growth factors (iFGFs) are key regulators of voltage-gated sodium channels. In heart failure (HF), the late sodium current (I NaL) is markedly enhanced and contributes to arrhythmogenic action potentials (APs). However, I NaL regulation by iFGFs and its therapeutic targeting in HF have not been previously studied. We aimed to investigate the expression of iFGF11-14 splice isoforms and tested for the effects of an iFGF12 A-mimicking inhibitory peptide (FixR) on I NaL magnitude and AP duration in rabbit and murine HF ventricular myocytes. We found that the expression of long iFGF isoforms (eg, iFGF12 A and iFGF13 S), which can inhibit I NaL, was markedly reduced both in a pressure/volume-overload induced HF rabbit model and in a transverse aortic constriction (TAC) induced murine HF model. I NaL density was significantly increased in HF myocytes, and importantly, cell pretreatment …

Biophysical Journal

Intracellular calcium (Ca 2+) is an essential mediator of normal cardiac function, and its dysregulation has been directly linked to atrial fibrillation (AF), the most common cardiac arrhythmia. Substantial sex differences exist in the prevalence, clinical representation, pathophysiology, therapy, and prognosis of AF, leading to less effective and sometimes harmful treatments for females. However, the associated mechanisms and causes remain largely unknown, partly due to female underrepresentation in both fundamental and clinical research. We recently revealed potential sex-specific mechanisms underlying the experimentally-observed increased female vs. male propensity for spontaneous Ca 2+ release events (SCRs), and this has important implications for understanding sex-specific AF pathophysiology and treatment. Here, we propose a multi-scale mechanistic modeling approach to determine the impact of sex …

bioRxiv

Background and AimsSubstantial sex-based differences have been reported in atrial fibrillation (AF), with female patients experiencing worse symptoms, increased complications from drug side effects or ablation, and elevated risk of AF-related stroke and mortality. Recent studies revealed sex-specific alterations in AF-associated Ca2+ dysregulation, whereby female cardiomyocytes more frequently exhibit potentially proarrhythmic Ca2+-driven instabilities compared to male cardiomyocytes. In this study, we aim to gain a mechanistic understanding of the Ca2+-handling disturbances and Ca2+-driven arrhythmogenic events in males vs females and establish their responses to Ca2+-targeted interventions.Methods and ResultsWe incorporated known sex differences and AF-associated changes in the expression and phosphorylation of key Ca2+-handling proteins and in ultrastructural properties and dimensions of atrial cardiomyocytes into our recently developed 3D atrial cardiomyocyte model that couples electrophysiology with spatially detailed Ca2+-handling processes. Our simulations of quiescent cardiomyocytes show increased incidence of Ca2+ sparks in female vs male myocytes in AF, in agreement with previous experimental reports. Additionally, our female model exhibited elevated propensity to develop pacing-induced spontaneous Ca2+ releases (SCRs) and augmented beat-to-beat variability in action potential (AP)-elicited Ca2+ transients compared with the male model. Parameter sensitivity analysis uncovered precise arrhythmogenic contributions of each component that was implicated in sex and/or AF alterations. Specifically …

Biophysical Journal

Experimental evidence points to greater diastolic Ca 2+(dCa) along with comparable sarcoplasmic reticulum Ca 2+ content in atrial cardiomyocytes from patients with atrial fibrillation (AF) compared with normal sinus rhythm (nSR). The augmented dCa may promote maladaptive Ca 2+-dependent signaling and arrhythmias. However, existing computational models incorporating all known components of AF-induced Ca 2+-handling remodeling (eg, increased ryanodine receptor Ca 2+ leak and Na+/Ca 2+ exchanger activity, reduced Ca 2+ influx via L-type Ca 2+ channels) consistently predict lower dCa for AF vs nSR conditions, contrasting the experimental findings. Mechanistically pinpointing the processes underlying the increased dCa may lead to the identification of novel therapeutic targets. Here, we performed computational modeling to gain quantitative understanding of possible ionic underpinnings of the …

Biophysical Journal

Currently, heart failure with preserved ejection fraction (HFpEF) is a human disease with increasing incidence, high mortality rates, and a narrow set of existing effective therapeutics. This disease has shown key sex-differences in which females present with higher prevalence, worse prognosis, and more severe diastolic dysfunction. The mechanisms that explain these differences are poorly understood, and this leads to inefficient drug development. Additionally, females are still underrepresented in both clinical trials and preclinical research. Many previous animal models of HFpEF failed to recapitulate key sex-differences of human HFpEF. Therefore, we proposed a preclinical HFpEF model that synergistically combines leptin receptor-deficient db/db mice with continuous 4-week aldosterone (Aldo) infusion. The HFpEF phenotype was confirmed using morphometry, echocardiography, and intracellular Ca imaging …

Biophysical Journal

386a Tuesday, February 13, 2024 expressed in the LM and ID, with low expression at the t-tubules. A similar pattern is observed for iFGF13, the predominant iFGF in the murine heart, and iFGF14. iFGF11 was not detected in murine myocytes, while iFGF12s were found throughout the cells but at a much lower expression. The expression of NaV1. 1 was evenly distributed to the t-tubules, while NaV1. 6 and NaV1. 8 were concentrated in the t-tubules close to the LM. These results suggest various subpopulations of NaV channel and iFGF complexes within each subcellular microdomain. In the future, we will study how different NaV channels and iFGFs colocalize and whether their interactions are altered in diseased cardiomyocytes.

Biophysical Journal

384a Tuesday, February 13, 2024 electrophysiological (EP) and structural remodeling. Though overall incidence of AF is similar between sexes, men develop AF at an earlier age compared to women, however women have poorer treatment outcomes and increased risk of mortality. The sex-specific mechanisms underlying these disparities is currently unclear and is further complicated by sparse knowledge about how the healthy atria differs between sexes. The objective of this study is to investigate inherent baseline sex differences in EP and arrhythmia susceptibility in the intact atria of a physiologically relevant animal model. Hearts were extracted from male and female New Zealand White rabbits (3-4 months) and the atria optically mapped using voltage (RH237) and Ca-sensitive (Rhod-2 AM) indicators. Electrical stimulation was performed via a pacing electrode on the right atrium. Baseline EP and arrhythmia …

Biophysical Journal

Atrial fibrillation (AF) is the most common arrhythmia worldwide. Though AF affects men and women at a similar rate there are major differences in epidemiology, manifestation, pathophysiology, treatment, and outcomes between sexes. Notably, men are more likely to be diagnosed with AF at an earlier age, and women are more likely to have worse AF symptoms that typically last longer and are more severe. Overall, this leads to a poorer quality of life and worse prognosis for women with AF compared to men. Studies investigating sex-related differences in the pathophysiology underlying AF have so far been limited, and thus the sex-specific mechanisms remain poorly understood. To address these knowledge gaps, we developed biophysically detailed mechanistic models of cardiac myocyte and tissue electrophysiology that integrate current knowledge of ion channel profiles and calcium (Ca) handling properties …

Biophysical Journal

1617-Pos Contribution of desmoplakin isoforms to desmosome architecture Krishna A. Patel, Collin M. Ainslie, Alexa L. Mattheyses. Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA. Desmosomes are cell-cell junctions that maintain the integrity of tissues that experience significant mechanical stress, such as the skin and heart. Desmosomal plaque proteins are connected to the intermediate filaments by desmoplakin (DP), and genetic variants in DP are associated with several skin and heart pathologies, including erythrokeratodermia and arrhythmogenic cardiomyopathy. DP contains three domains: an N-terminal plakin head, a central rod, and an intermediate filament-binding C-terminal tail. Furthermore, DP has three splice isoforms (DPI, DPIa, and DPII) that differ only in the length of the rod domain and have distinct tissue-specific expression …

Biophysical Journal

The use of fast in silico prediction methods for protein-ligand binding free energies holds significant promise for the initial phases of drug development. Numerous traditional physics-based models (e.g., implicit solvent models), however, tend to either neglect or heavily approximate entropic contributions to binding due to their computational complexity. Consequently, such methods often yield imprecise assessments of binding strength. Machine learning models provide accurate predictions and can often outperform physics-based models. They, however, are often prone to overfitting, and the interpretation of their results can be difficult. Physics-guided machine learning models combine the consistency of physics-based models with the accuracy of modern data-driven algorithms. This work integrates physics-based model conformational entropies into a graph convolutional network. We introduce a new neural …

Biophysical Journal

Dipalmitoylphosphatidylcholine (DPPC) is a common model system for exploring the phase behavior of Lung Surfactant-the lipid-protein layer coating of the alveolar interface that facilitates breathing by reducing the line tension. The availability of enantiomers of DPPC in purified form allows us to investigate the chiral nature of model lipid monolayers through fluorescence microscopy and traditional Langmuir thermodynamic techniques. We focus on mixtures of DPPC with cholesterol, hexadecanol (HD), and palmitic acid (PA), which have been previously studied and shown to form equilibrium morphologies over experimental time scales REF: Valtierrez et al., Sci. Adv. v: 8: 14, 2022). In this poster, we will assess the use of complementary image processing and analysis routines to measure the curvature of morphologies within these monolayers. The introduction of cholesterol within the monolayers stabilizes …

Biophysical Journal

450a Wednesday, February 14, 2024 applicability of our platform by studying a range of cell-derived native vesicles, which includes bacterial outer membrane vesicles, mammalian plasma membrane-derived GPMVs, ER-derived microsomes, and other physiological membrane vesicles. In each of these cases, the application of our platform enables direct detection and ID of endogenous integral and peripheral MP-complexes, along with the bound ligands and lipids. Applying this platform, we demonstrate our ability to screen ligands against specific bacterial MP-complexes that are key targets for developing antibiotics, directly from their native membrane, at their endogenous level of expression. In a separate application, we show how specific post-translational modifications regulate molecular assemblies of neuronal SNARE complexes. Together, we present a broadly applicable platform to study the molecular …

Biophysical Journal

1Department of Cell Biology, Yale University, New Haven, CT, USA, 2Department of Pharmacology, Yale University, New Haven, CT, USA. The local membrane environment plays a paramount role in regulating the biology of membrane proteins (MP). Standard detergent-based MP-solubilization strategies disrupt the native bilayer-environment, leading to a fundamental gap in our understanding of how nanoscale molecular organization of MPs regulates their ability to elicit cellular responses. Addressing this, we have developed a proteome-wide quantitative guide for high-throughput, spatiallyresolved extraction of MPs into tunable native nanodiscs of 8-20 nm diameter. This enables downstream structural/functional studies of target MPs directly from native membranes. Combining existing MAPs with in-house chaincontrolled polymers, we developed a high-throughput, fluorescence-based assay for rapid screening …

Biophysical Journal

The organization of membrane proteins (MP) and lipids in the cell membrane is vital for all living organisms. To study this, we have developed a novel tunable in-vitro native mass spectrometry (nMS) platform that allows direct detection of the organization of MPs and bound lipids from membranes. This method employs customizable lipid bilayers to quantitatively reveal how specific membrane compositions and biophysical properties, such as curvature, tension, and fluidity, influence the lipid binding and functional arrangement of MPs. Here, we apply this platform to understand the lipid specificity of a trafficking MP, VAMP2 across the organellar membrane and how that tunes its biological function. Following its trafficking pathway, we study VAMP2 directly from liposomes mimicking different organelle membranes encountered during its trafficking pathways, such as ER, Golgi, synaptic vesicle (SV), and PM. By directly …

Biophysical Journal

Desmosomes are junctional protein assemblies found in epithelial and cardiac tissue that assist in maintaining tissue plasticity and integrity. They are composed of transmembrane cadherins desmocollin and desmoglein and intracellular plaque proteins plakoglobin (PG), plakophilin (PKP), and desmoplakin (DP). DP consists of a C-terminal tail domain that anchors the desmosome to intermediate filaments, central rod domain, and N-terminal head that binds PG and PKP. Mutations in desmosomal proteins result in weakened cell-cell adhesion in skin and heart diseases. Desmosomes exist in two states, calcium-dependent and hyper-adhesive, and can modulate between the two in dynamic processes such as wound healing. However, it is unclear how desmosome architecture is altered in hyper-adhesion and how the architecture impacts desmosome function. Hyper-adhesion can be conferred through different …

Biophysical Journal

Desmosomes are cell-cell junctions that maintain the integrity of tissues that experience significant mechanical stress, such as the skin and heart. Desmosomal plaque proteins are connected to the intermediate filaments by desmoplakin (DP), and genetic variants in DP are associated with several skin and heart pathologies, including erythrokeratodermia and arrhythmogenic cardiomyopathy. DP contains three domains: an N-terminal plakin head, a central rod, and an intermediate filament-binding C-terminal tail. Furthermore, DP has three splice isoforms (DPI, DPIa, and DPII) that differ only in the length of the rod domain and have distinct tissue-specific expression patterns. DPI is the primary cardiac isoform and is present in all desmosomes, whereas DPIa and DPII are expressed in stratified epithelia. Although DP’s binding partners and spliced isoforms are known, their spatial arrangement and functional …

Biophysical Journal

484a Wednesday, February 14, 2024 and efficient method for measuring surface-bound molecule flexibility. This method involves attaching a fluorophore to the molecule’s end and monitoring emitted light polarization as the molecule fluctuates. Using confocal polarization microscopy, we track the average changes in orientation of the fluorophore, which serves as a proxy for the flexibility of labeled molecules. Rigid molecules display minimal changes in fluorophore orientation, whereas flexible ones exhibit more significant changes in orientation. Using this molecular flexibility measurement technique, we first conducted in vitro experiments using DNA oligos attached to giant unilamellar vesicles. We find that increasing the length of the oligo increases its measured flexibility, consistent with computational models of a fluctuating polymer attached to a surface. Next, we investigated the role of surrounding molecules …

Biophysical Journal

Podosomes are punctate actin-rich cytoskeletal structures used by dendritic cells and macrophages to sense the mechanical properties of the surrounding extracellular environment, helping them patrol for pathogens. Podosomes are sensitive to substrate stiffness and probe the extra-cellular matrix by polymerizing actin from the cap against the plasma membrane, exerting force onto the substrate and detecting the opposing forces. Cycles of polymerization and collapse result in height oscillations of the podosome core. Interestingly, podosomes form clusters that exhibit collective oscillatory behavior, resulting in podosome height waves in which a local group of podosomes sequentially increase and then decrease their F-actin content. A model to explain the emergence of such collective behavior was missing until now, and no method had been able to quantify the spatiotemporal propagation of collective podosome …

Biophysical Journal

Many cell functions require a concerted effort from multiple membrane proteins, for example, for signaling, cell division, and endocytosis. One contribution to their successful self-organization stems from the membrane deformations that these proteins induce. While the pairwise interaction potential of two membrane-deforming spheres has recently been measured, membrane-deformation-induced interactions have been predicted to be nonadditive, and hence their collective behavior cannot be deduced from this measurement. We here employ a colloidal model system consisting of adhesive spheres and giant unilamellar vesicles to test these predictions by measuring the interaction potential of the simplest case of three membrane-deforming, spherical particles. We quantify their interactions and arrangements and, for the first time, experimentally confirm and quantify the nonadditive nature of membrane-deformation …

Biophysical Journal

Comparative methods in molecular evolution and structural biology rely heavily upon the site-wise analysis of DNA sequence and protein structure, both static forms of information. However, it is widely accepted that protein function results from nanoscale nonrandom machine-like motions induced by evolutionarily conserved molecular interactions. Comparisons of molecular dynamics (MD) simulations conducted between homologous sites representative of different functional or mutational states can potentially identify local effects on binding interaction and protein evolution. In addition, comparisons of different (i.e., nonhomologous) sites within MD simulations could be employed to identify functional shifts in local time-coordinated dynamics indicative of logic gating within proteins. However, comparative MD analysis is challenged by the large fraction of protein motion caused by random thermal noise in the …

Biophysical Journal

Mechanotransduction, the conversion of mechanical forces into biochemical signals, is essential for tissue development and physiological maintenance. Drosophila embryogenesis is ideal to study mechanotransduction given the significant strain cells experience to drive tissue-scale deformations. We aimed to understand the relationship between mechanical strain and Ca 2+ spiking in epithelial cells during Drosophila gastrulation. Previous research highlights the role of mechanosensitive ion channels (MSCs) in facilitating Ca 2+-dependent mechanotransduction during the processes of egg activation and dorsal closure. Intrigued by these reports, we examined the frequency and distribution of Ca 2+ spikes during the mechanically intensive process of gastrulation. We discover dynamic Ca 2+ spiking patterns during gastrulation that notably colocalize with high-strain areas. Fluorescence, time-lapse microscopy …

Biophysical Journal

The N-terminal domain of the Huntingtin protein (htt), called N17, plays a crucial role in the physiology of Huntingtonʼs disease. N17 forms an amphipathic helix when in contact with membranes or in aggregate form. Studies have indicated that N17 is responsible for the localization of htt to various organelles in the cell, including the endoplasmic reticulum, mitochondria and Golgi apparatus. It has also been implicated in increasing the rate of htt aggregation through its tendency to self interact. Computational and experimental techniques have delineated a detailed picture of the interactions of N17 with planar membranes. However, recent reports have suggested that N17 has an affinity for curved membranes. Given the uneven and curved shapes of organelles, it is important to examine the mechanistic preferences that drive the preferential partitioning of N17 to curved membranes. We used coarse-grained …

Biophysical Journal

Many forms of biological matter exhibit similar water-responsive changes in their size and mechanical properties. Recent experiments on bacterial spores led to the formulation of a theory of the mechanics of the spores based on the dominant role of hydration forces [1]. The theory was particularly successful in predicting unusual mechanical properties including a strong nonlinear elasticity and a jamming-driven transition in mechanical properties at short timescales, both of which were confirmed by atomic force microscopy experiments on spores. The simplicity of the underlying assumptions of the theory suggests that the theory could be applicable to other types of biological matter that exhibit water-responsive behavior. Here we present atomic force microscopy measurements on cellulose-based materials that reproduced the strong nonlinear elasticity discovered originally with spores. Quantitative analysis of the …

Biophysical Journal

Proteins are molecular machines and to understand how they work, we need to understand how they move. New pump-probe time-resolved X-ray diffraction methods open up ways to initiate and observe protein motions with atomistic detail in crystals on biologically relevant timescales. However, the practical limitations of these experiments demand parallel development of effective molecular dynamics approaches to accelerate progress and extract meaning. Here, we establish robust and accurate methods for simulating dynamics in protein crystals, a nontrivial process requiring careful attention to equilibration, environmental composition, and choice of force fields. With more than seven milliseconds of sampling of a single chain, we identify critical factors controlling agreement between simulation and experiments and show that simulated motions recapitulate ligand-induced conformational changes. This work …

Biophysical Journal

Electric-field stimulated X-ray crystallography (EF-X) involves applying an electric-field pulse to a protein crystal and using time-resolved X-ray diffraction to observe the response to the applied field. In this way, the dynamics of proteins can be resolved with high spatial resolution on a sub-microsecond timescale, making it highly suitable for comparison with molecular dynamics (MD) simulations. We emulate the EF-X experiment using all-atom MD simulations of a protein crystal with an applied electric field. As a model system, we simulate a 3× 3× 3 supercell of a PDZ domain and an electric field of 1 MV/cm. We compare the consistency of the induced motions in simulation and experiment. The EF-X experiment provides a rich data set and a novel approach to validating MD simulation.

Biophysical Journal

The blood-brain barrier is a highly complex physiological barrier that separates the blood from the central nervous system to maintain the latter's biological equilibrium. LDL receptor-related protein 1 (LRP1) is a receptor involved in BBB transcytosis and can be used by physiological or artificially induced processes. LRP1 is critical for the trafficking of misfolded proteins such as amyloid β, hyper-phosphorylated tau, and α-synuclein. Understanding its structure and function is essential to fully understanding neurological diseases like Alzheimer's, Parkinson's, Huntington's, and other related dementias. LRP1 is a modular membrane protein composed of 4544 amino acids, around 1200 of which are involved in three long and flexible structures that contain coordinated calcium ions and are decorated with small sugar chains called glycans. These three flexible components are believed to have an active role in ligand …

Biophysical Journal

Porphyromonas gingivalis is a keystone pathogen of chronic periodontitis. During infection, P. gingivalis evades the host immune system via a number of virulence factors, such as the PG0352 sialidase. Several reports implicate sialidases in bacterial complement resistance but the underlying molecular mechanism has been elusive. Many complement factors and regulatory proteins are sialylated (modified with glycans containing terminal sialic acids), which is essential to their function. We provide functional and structural evidence that PG0352 sialidase can disarm the complement system via desialylation of complement proteins and regulators. Biochemical analyses reveal PG0352 can desialylate human serum and complement factors, thereby protecting bacteria from serum killing, mediated by membrane attack complement (MAC) complex formation. Structural studies reveal PG0352 is a dual domain sialidase …

Biophysical Journal

Seminal fluid in humans contain approximately 1-3 mM zinc, more than 100-fold higher than the amount circulating throughout the human body. Zinc is both essential and the most abundant trace metal in the body; however, there is no clear understanding of why zinc is enriched in human semen. We hypothesized that this abundant zinc may influence the physiology of sperm between mating and fertilization. Most of the zinc in seminal fluid is bound to proteins; thus, using the fluorescent zinc indicator FluoZin-1 to quantify the readily available zinc in seminal fluid, we found that pig semen includes 9.6±0.4 μM labile zinc (N= 8), and 5.0±0.7 μM for human (N= 3). To determine whether seminal zinc is imported by mammalian sperm, we used the intracellular zinc indicator FluoZin3-AM to observe changes before and after application of zinc. We found that mouse sperm quickly import extracellular zinc in a concentration …