The 100 most-cited articles in Biophysical Journal, 2024

ABSTRACT Liquid-liquid phase separation (LLPS) is thought to be a main driving force in the formation of membraneless organelles. Examples of such organelles include the centrosome, central spindle, and stress granules. Recently, it has been shown that coiled-coil (CC) proteins, such as the centrosomal proteins pericentrin, spd-5, and centrosomin, might be capable of LLPS. CC domains have physical features that could make them the drivers of LLPS, but it is unknown if they play a direct role in the process. We developed a coarse-grained simulation framework for investigating the LLPS propensity of CC proteins, in which interactions which support LLPS arise solely from CC domains. We show, using this framework, that the physical features of CC domains are sufficient to drive LLPS of proteins. The framework is specifically designed to investigate how the number of CC domains, as well as multimerization state of CC domains, can affect LLPS. We show that small model proteins with as few as two CC domains can phase separate. Increasing the number of CC domains up to four per protein can…

ABSTRACT Protein post-translational modifications, such as phosphorylation, are important regulatory signals for diverse cellular functions. In particular, intrinsically disordered protein regions (IDRs) are subject to phosphorylation as a means to modulate their interactions and functions. Toward understanding the relationship between phosphorylation in IDRs and specific functional outcomes, we must consider how phosphorylation affects the IDR conformational ensemble. Various experimental techniques are suited to interrogate the features of IDR ensembles; molecular simulations can provide complementary insights and even illuminate ensemble features that may be experimentally inaccessible. Therefore, we sought to expand the tools available to study phosphorylated IDRs by all-atom Monte Carlo simulations. To this end, we implemented parameters for phosphoserine (pSer) and phosphothreonine (pThr) into the OPLS version of the continuum solvent model, ABSINTH, and assessed their performance in all-atom simulations compared to published findings. We simulated short (< 20 residues) and…

Sensing of the biophysical properties of membranes using molecular reporters has recently regained widespread attention. This was elicited by the development of new probes of exquisite optical properties and increased performance, combined with developments in fluorescence detection. Here, we report on fluorescence lifetime imaging of various rigid and flexible fluorescent dyes to probe the biophysical properties of synthetic and biological membranes at steady state as well as upon the action of external membrane-modifying agents. We tested the solvatochromic dyes Nile red and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD), the viscosity sensor Bodipy C 12 , the flipper dye FliptR, as well as the dyes 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), Bodipy C 16 , lissamine-rhodamine, and Atto647, which are dyes with no previous reported environmental sensitivity. The performance of the fluorescent probes, many of which are commercially available, was benchmarked with well-known environmental reporters, with Nile red and…

Trypsin is a very common enzyme used in cell culture to harvest cells by cleaving the proteins responsible for cell adhesion. However, trypsin also induces undesirable effects on cells, such as altering membrane proteins and the cytoskeleton, changing the composition of the cytoplasm and the cell volume, and even leading to cell death when used improperly. Using attenuated total reflection in the terahertz domain, confocal microscopy, and the propidium iodide test, we quantified in real time the change in cytoplasmic content induced by trypsin proteolysis on Madin-Darby canine kidney epithelial cells. We have observed a cytoplasmic modification from the very first seconds of trypsinization, following the change of cell volume due to mechanical re-equilibrium of the membrane. We found that the cytoplasmic alteration is associated with a transfer of small solutes: electrolytes and metabolites. We also found a very good nonlinear correlation between the side effects monitored by terahertz sensing and the cell height, regardless of the dependence of the cell height on trypsin…

The adaptive immune response is orchestrated by just two cell types, T cells and B cells. Both cells possess the remarkable ability to recognize virtually any antigen through their respective antigen receptors-the T cell receptor (TCR) and B cell receptor (BCR). Despite extensive investigations into the biochemical signaling events triggered by antigen recognition in these cells, our ability to predict or control the outcome of T and B cell activation remains elusive. This challenge is compounded by the sensitivity of T and B cells to the biophysical properties of antigens and the cells presenting them-a phenomenon we are just beginning to understand. Recent insights underscore the central role of mechanical forces in this process, governing the conformation, signaling activity, and spatial organization of TCRs and BCRs within the cell membrane, ultimately eliciting distinct cellular responses. Traditionally, T cells and B cells have been studied independently, with researchers working in parallel to decipher the mechanisms of activation. While these investigations have unveiled…

Phosphorylated TAR DNA-binding protein of 43 kDa (TDP-43) is present within the aggregates of several age-related neurodegenerative disorders, such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease, to the point that the presence of phosphorylated TDP-43 is considered a hallmark of some of these diseases. The majority of known TDP-43 phosphorylation sites detected in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients is located in the low-complexity domain (LCD), the same domain that has been shown to be critical for TDP-43 liquid-liquid phase separation (LLPS). However, the effect of these LCD phosphorylation sites on TDP-43 LLPS has been largely unexplored, and any work that has been done has mainly focused on sites near the C-terminal end of the LCD. Here, we used a phosphomimetic approach to explore the impact of phosphorylation at residues S332 and S333, sites located within the transiently α-helical region of TDP-43 that have been observed to be phosphorylated in disease, on protein LLPS. Our turbidimetry and…

Lipid membranes play a crucial role in regulating the body's water balance by adjusting their properties in response to hydration. The intercellular lipid matrix of the stratum corneum (SC), the outermost skin layer, serves as the body's primary defense against environmental factors. Osmolytes, including urocanic acid (UCA) and glycerol, are key components of the natural moisturizing factor that help the SC resist osmotic stress from dry environments. This study examines the effects of UCA and glycerol (each at 5 mol %) on isolated human SC lipids. For this, different techniques were employed, offering complementary information of the system's multiscale characteristics, including humidity-scanning quartz crystal microbalance with dissipation monitoring, infrared spectroscopy, x-ray diffraction, electrical impedance spectroscopy, and studies of water loss and permeability. Our results show that UCA increases water sorption and makes lipid films more liquid-like at high relative humidity, without significantly altering the lipid lamellar structure, chain order, or orthorhombic chain…

Many proteins with intrinsically disordered regions undergo liquid-liquid phase separation under specific conditions in vitro and in vivo. These complex biopolymers form a metastable phase with distinct mechanical properties defining the timescale of their biological functions. However, determining these properties is nontrivial, even in vitro, and often requires multiple techniques. Here we report the measurement of both viscosity and surface tension of biomolecular condensates via correlative fluorescence microscopy and atomic force microscopy (AFM) in a single experiment (fluorescence recovery after probe-induced dewetting, FRAP-ID). Upon surface tension evaluation via regular AFM-force spectroscopy, controlled AFM indentations induce dry spots in fluorescent condensates on a glass coverslip. The subsequent rewetting exhibits a contact line velocity that is used to quantify the condensed-phase viscosity. Therefore, in contrast with fluorescence recovery after photobleaching (FRAP), where molecular diffusion is observed, in FRAP-ID fluorescence recovery is obtained through fluid…

Stochastic models of gene expression are typically formulated using the chemical master equation, which can be solved exactly or approximately using a repertoire of analytical methods. Here, we provide a tutorial review of an alternative approach based on queueing theory that has rarely been used in the literature of gene expression. We discuss the interpretation of six types of infinite-server queues from the angle of stochastic single-cell biology and provide analytical expressions for the stationary and nonstationary distributions and/or moments of mRNA/protein numbers and bounds on the Fano factor. This approach may enable the solution of complex models that have hitherto evaded analytical solution.

Abstract Biological condensates often emerge as a multi-droplet state and never coalesce into one large droplet within the experimental timespan. Previous work revealed that the sticker-spacer architecture of biopolymers may dynamically stabilize the multi-droplet state. Here, we simulate the condensate coalescence using metadynamics approach and reveal two distinct physical mechanisms underlying the fusion of droplets. Condensates made of sticker-spacer polymers readily undergo a kinetic arrest when stickers exhibit slow exchange while fast exchanging stickers at similar levels of saturation allow merger to equilibrium states. On the other hand, condensates composed of homopolymers fuse readily until they reach a threshold density. Increase in entropy upon inter-condensate mixing of chains drives the fusion of sticker-spacer chains. We map the range of mechanisms of kinetic arrest from slow sticker exchange dynamics to density mediated in terms of energetic separation of stickers and spacers. Our predictions appear to be in qualitative agreement with recent experiments probing…

Plasma membrane fluidity is an important phenotypic feature that regulates the diffusion, function, and folding of transmembrane and membrane-associated proteins. In bacterial cells, variations in membrane fluidity are known to affect respiration, transport, and antibiotic resistance. Membrane fluidity must therefore be tightly regulated to adapt to environmental variations and stresses such as temperature fluctuations or osmotic shocks. Quantitative investigation of bacterial membrane fluidity has been, however, limited due to the lack of available tools, primarily due to the small size and membrane curvature of bacteria that preclude most conventional analysis methods used in eukaryotes. Here, we develop an assay based on total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) to directly measure membrane fluidity in live bacteria via the diffusivity of fluorescent membrane markers. With simulations validated by experiments, we could determine how the small size, high curvature, and geometry of bacteria affect diffusion measurements and correct subsequent…

We present a method to differentiate organisms solely by their motion based on the generalized Langevin equation (GLE) and use it to distinguish two different swimming modes of strongly confined unicellular microalgae Chlamydomonas reinhardtii. The GLE is a general model for active or passive motion of organisms and particles that can be derived from a time-dependent general many-body Hamiltonian and in particular includes non-Markovian effects (i.e., the trajectory memory of its past). We extract all GLE parameters from individual cell trajectories and perform an unbiased cluster analysis to group them into different classes. For the specific cell population employed in the experiments, the GLE-based assignment into the two different swimming modes works perfectly, as checked by control experiments. The classification and sorting of single cells and organisms is important in different areas; our method, which is based on motion trajectories, offers wide-ranging applications in biology and medicine.

Plant development relies on the precise coordination of cell growth, which is influenced by the mechanical constraints imposed by rigid cell walls. The hormone auxin plays a crucial role in regulating this growth by altering the mechanical properties of cell walls. During the postembryonic formation of lateral roots, pericycle cells deep within the main root are triggered by auxin to resume growth and divide to form a new root. This growth involves a complex interplay between auxin, growth, and the resolution of mechanical conflicts with the overlying endodermis. However, the exact mechanisms by which this coordination is achieved are still unknown. Here, we propose a model that integrates tissue mechanics and auxin transport, revealing a connection between the auxin-induced relaxation of mechanical stress in the pericycle and auxin signaling in the endodermis. We show that the endodermis initially limits the growth of pericycle cells, resulting in a modest initial expansion. However, the associated stress relaxation is sufficient to redirect auxin to the overlying endodermis, which…

Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured…

ABSTRACT The asymmetry of membranes has a significant impact on their biophysical characteristics and behavior. This study investigates the composition and mechanical properties of symmetric and asymmetric membranes in giant unilamellar vesicles (GUVs) made of phosphatidylcholine (POPC) and phosphatidic acid (POPA). A combination of fluorescence quantification, zeta potential measurements, micropipette aspiration and bilayer molecular dynamics simulations are used to characterize these membranes. The outer leaflet composition in vesicles is found consistent across the two preparation methods we employed, namely electroformation and inverted emulsion transfer. However, characterizing the inner leaflet poses challenges. Micropipette aspiration of GUVs show that oil residues do not substantially alter membrane elasticity, but simulations reveal increased membrane thickness and decreased interleaflet coupling in the presence of oil. Asymmetric membranes with a POPC:POPA mixture in the outer leaflet and POPC in the inner leaflet display similar stretching elasticity values to symmetric…

Compaction is the first morphogenetic movement of the eutherian mammals and involves a developmentally regulated adhesion process. Previous studies investigated cellular and mechanical aspects of compaction. During mouse and human compaction, cells spread onto each other as a result of a contractility-mediated increase in surface tension pulling at the edges of their cell-cell contacts. However, how compaction may affect the mechanical stability of cell-cell contacts remains unknown. Here, we used a dual pipette aspiration assay on cell doublets to quantitatively analyze the mechanical stability of compacting mouse embryos. We measured increased mechanical stability of contacts with rupture forces growing from 40 to 70 nN, which was highly correlated with cell-cell contact expansion. Analyzing the dynamic molecular reorganization of cell-cell contacts, we find minimal recruitment of the cell-cell adhesion molecule Cdh1 (also known as E-cadherin) to contacts but we observe its reorganization into a peripheral adhesive ring. However, this reorganization is not associated with…

We present phalloidin-based points accumulation for imaging in nanoscale topography (phalloidin-PAINT), enabling quantitative superresolution imaging of filamentous actin (F-actin) in the cell body and delicate membrane protrusions. We demonstrate that the intrinsic phalloidin dissociation enables PAINT superresolution microscopy in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-PAINT are its ability to consistently quantify F-actin at the nanoscale throughout the entire cell and its enhanced preservation of fragile cellular structures. In a proof-of-concept study, we employed phalloidin-PAINT to superresolve F-actin structures in U2OS and dendritic cells (DCs). We demonstrate more consistent F-actin quantification in the cell body and structurally delicate membrane protrusions of DCs compared with direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse DCs as the model system, we show F-actin…

Collective cell invasion (CCI), a canon of most invasive solid tumors, is an emergent property of the interactions between cancer cells and their surrounding extracellular matrix (ECM). However, tumor populations invariably consist of cells expressing variable levels of adhesive proteins that mediate such interactions, disallowing an intuitive understanding of how tumor invasiveness at a multicellular scale is influenced by spatial heterogeneity of cell-cell and cell-ECM adhesion. Here, we have used a Cellular Potts model-based multiscale computational framework that is constructed on the histopathological principles of glandular cancers. In earlier efforts on homogenous cancer cell populations, this framework revealed the relative ranges of interactions, including cell-cell and cell-ECM adhesion that drove collective, dispersed, and mixed multimodal invasion. Here, we constitute a tumor core of two separate cell subsets showing distinct intra- and inter-subset cell-cell or cell-ECM adhesion strengths. These two subsets of cells are arranged to varying extents of spatial…

Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in immortalized human astrocytes and GBM cells via fluorescence microscopy and image analysis techniques. Results from our study show that…

COG0523 proteins, also known as nucleotide-dependent metallochaperones, are a poorly understood class of small P-loop G3E GTPases. Multiple family members play critical roles in bacterial pathogen survival during an infection as part of the adaptive response to host-mediated "nutritional immunity." Our understanding of the structure, dynamics, and molecular-level function of COG0523 proteins, apart from the eukaryotic homolog, Zng1, remains in its infancy. Here, we use X-ray absorption spectroscopy to establish that Acinetobacter baumannii ( Ab ) ZigA coordinates Zn II using all three cysteines derived from the invariant CXCC motif to form an S 3 (N/O) coordination complex, a feature inconsistent with the Zn II -bound crystal structure of a distantly related COG0523 protein of unknown function from Escherichia coli , Ec YjiA. The binding of Zn II and guanine nucleotides is thermodynamically linked in Ab ZigA, and this linkage is more favorable for the substrate GTP relative to the product GDP. Part of this coupling originates with nucleotide-induced stabilization of the G-domain…

The transport of molecules across cell membranes is vital for proper cell function and effective drug delivery. While most cell membranes naturally possess an asymmetric lipid composition, research on membrane transport predominantly uses symmetric lipid membranes. The permeation through the asymmetric membrane is then calculated as a sum of the inverse permeabilities of leaflets from symmetric bilayers. In this study, we examined how two types of amphiphilic molecules translocate across both asymmetric and symmetric membranes. Using computer simulations with both coarse-grained and atomistic force fields, we calculated the free energy profiles for the passage of model amphiphilic peptides and a lipid across various membranes. Our results consistently demonstrate that while the free energy profiles for asymmetric membranes with a small differential stress concur with symmetric ones in the region of lipid headgroups, the profiles differ around the center of the membrane. In this region, the free energy for the asymmetric membrane transitions between the profiles for two symmetric…

It is important to understand the behaviors of fluorescent molecules because, firstly, they are often utilized as probes in biophysical experiments and, secondly, they are crucial cofactors in biological processes such as photosynthesis. A phenomenon called "fluorescence quenching" occurs when fluorophores are present at high concentrations, but the mechanisms for quenching are debated. Here, we used a technique called "in-membrane electrophoresis" to generate concentration gradients of fluorophores within a supported lipid bilayer, across which quenching was expected to occur. Fluorescence lifetime imaging microscopy (FLIM) provides images where the fluorescence intensity in each pixel is correlated to fluorescence lifetime: the intensity provides information about the location and concentration of fluorophores and the lifetime reveals the occurrence of energy-dissipative processes. FLIM was used to compare the quenching behavior of three commonly used fluorophores: Texas Red (TR), nitrobenzoaxadiazole (NBD), and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). FLIM images…

Biomolecular condensates have emerged as a powerful new paradigm in cell biology with broad implications to human health and disease, particularly in the nucleus where phase separation is thought to underly elements of chromatin organization and regulation. Specifically, it has been recently reported that phase separation of heterochromatin protein 1alpha (HP1α) with DNA contributes to the formation of condensed chromatin states. HP1α localization to heterochromatic regions is mediated by its binding to specific repressive marks on the tail of histone H3, such as trimethylated lysine 9 on histone H3 (H3K9me3). However, whether epigenetic marks play an active role in modulating the material properties of HP1α and dictating emergent functions of its condensates remains to be understood. Here, we leverage a reductionist system, composed of modified and unmodified histone H3 peptides, HP1α, and DNA, to examine the contribution of specific epigenetic marks to phase behavior of HP1α. We show that the presence of histone peptides bearing the repressive H3K9me3 is compatible with HP1α…

Understanding cell fate decision-making during complex biological processes is an open challenge that is now aided by high-resolution single-cell sequencing technologies. Specifically, it remains challenging to identify and characterize transition states corresponding to "tipping points" whereby cells commit to new cell states. Here, we present a computational method that takes advantage of single-cell transcriptomics data to infer the stability and gene regulatory networks (GRNs) along cell lineages. Our method uses the unspliced and spliced counts from single-cell RNA sequencing data and cell ordering along lineage trajectories to train an RNA splicing multivariate model, from which cell-state stability along the lineage is inferred based on spectral analysis of the model's Jacobian matrix. Moreover, the model infers the RNA cross-species interactions resulting in GRNs and their variation along the cell lineage. When applied to epithelial-mesenchymal transition in ovarian and lung cancer-derived cell lines, our model predicts a saddle-node transition between the epithelial and…

Many bacterial habitats—ranging from gels and tissues in the body to cell-secreted exopolysaccharides in biofilms—are rheologically complex, undergo dynamic external forcing, and have unevenly distributed nutrients. How do these features jointly influence how the resident cells grow and proliferate? Here, we address this question by studying the growth of Escherichia coli dispersed in granular hydrogel matrices with defined and highly tunable structural and rheological properties, under different amounts of external forcing imposed by mechanical shaking, and in both aerobic and anaerobic conditions. Our experiments establish a general principle: that the balance between the yield stress of the environment that the cells inhabit, σ y , and the external stress imposed on the environment, σ , modulates bacterial growth by altering transport of essential nutrients to the cells. In particular, when σ y < σ , the environment is easily fluidized and mixed over large scales, providing nutrients to the cells and sustaining complete cellular growth. By contrast, when σ y > σ , the elasticity…

Intracellular membrane traffic involves controlled membrane fission and fusion and is essential for eukaryotic cell homeostasis. Most intracellular fusion is facilitated by Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, which catalyze membrane merging by assembly of a coiled helical bundle of four 60- to 70-residue "SNARE motifs." Perhaps no intracellular fusion reaction is as tightly regulated as that at the neuronal synapse, mediated by the synaptic vesicle SNARE Synaptobrevin-2 and the presynaptic plasma membrane SNAREs Syntaxin-1a and SNAP25. SNAP25 is different from its partner SNAREs: it contributes not one but two SNARE motifs to the final complex and instead of transmembrane domains is anchored in the membrane by post-translational palmitoylation of a long flexible linker between the SNARE motifs. Despite reports of structural and functional differences between the two SNARE motifs, many models of SNARE assembly and fusion consider SNAP25 to be a single functional unit and do not address how linking two distinct motifs in a single…

Lipid compositional asymmetry across the leaflets of the plasma membrane is an ubiquitous feature in eukaryotic cells. How this asymmetry is maintained is thought to be primarily controlled by active transport of lipids between leaflets. This strategy is facilitated by the fact that long-tail phospholipids and sphingolipids diffuse through the lipid bilayer slowly-taking many hours or days. However, a lipid like cholesterol-which is the most abundant lipid in the plasma membrane of animal cells-has been harder to pinpoint in terms of its favored side. In this work we show that, when a saturated lipid is added to a mix of the unsaturated lipid palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesterol, both cholesterol and the long-tail phospholipids organize asymmetrically across the membrane's leaflets naturally. In these extruded unilamellar vesicles, most cholesterol as well as the saturated lipid-dipalmitoylphosphatidylcholine or sphingomyelin-segregated to the inner leaflet while POPC preferentially localized in the outer leaflet. This asymmetric arrangement generated a slight…

The length-dependent activation (LDA) of maximum force and calcium sensitivity are established features of cardiac muscle contraction but the dominant underlying mechanisms remain to be fully clarified. Alongside the well-documented regulation of contraction via the thin filaments, experiments have identified an additional force-dependent thick-filament activation, whereby myosin heads parked in a so-called off state become available to generate force. This process produces a feedback effect that may potentially drive LDA. Using biomechanical modeling of a human left-ventricular myocyte, this study investigates the extent to which the off-state dynamics could, by itself, plausibly account for LDA, depending on the specific mathematical formulation of the feedback. We hypothesized four different models of the off-state regulatory feedback based on (A) total force, (B) active force, (C) sarcomere strain, and (D) passive force. We tested if these models could reproduce the isometric steady-state and dynamic LDA features predicted by an earlier published model of a human left-ventricle…

Solutions of some proteins phase separate into a condensed state of high protein concentration and a dispersed state of low concentration. Such behavior is observed in living cells for a number of RNA-binding proteins that feature intrinsically disordered domains. It is relevant for cell function via the formation of membraneless organelles and transcriptional condensates. On a basic level, the process can be studied in vitro on protein domains that are necessary and sufficient for liquid-liquid phase separation (LLPS). We have performed distance distribution measurements by electron paramagnetic resonance for 13 sections in an N-terminal domain (NTD) construct of the protein fused in sarcoma (FUS), consisting of the QGSY-rich domain and the RGG1 domain, in the denatured, dispersed, and condensed state. Using 10 distance distribution restraints for ensemble modeling and three such restraints for model validation, we have found that FUS NTD behaves as a random-coil polymer under good-solvent conditions in both the dispersed and condensed state. Conformation distribution in the…

The inner lining of blood vessels, the endothelium, is made up of endothelial cells. Vascular endothelial (VE)-cadherin protein forms a bond with VE-cadherin from neighboring cells to determine the size of gaps between the cells and thereby regulate the size of particles that can cross the endothelium. Chemical cues such as thrombin, along with mechanical properties of the cell and extracellular matrix are known to affect the permeability of endothelial cells. Abnormal permeability is found in patients suffering from diseases including cardiovascular diseases, cancer, and COVID-19. Even though some of the regulatory mechanisms affecting endothelial permeability are well studied, details of how several mechanical and chemical stimuli acting simultaneously affect endothelial permeability are not yet understood. In this article, we present a continuum-level mechanical modeling framework to study the highly dynamic nature of the VE-cadherin bonds. Taking inspiration from the catch-slip behavior that VE-cadherin complexes are known to exhibit, we model the VE-cadherin homophilic bond as…

Cells can dynamically organize reactions through the formation of biomolecular condensates. These viscoelastic networks exhibit complex material properties and mesoscale architectures, including the ability to form multiphase assemblies. It was shown previously that condensates with complex architectures may arise at equilibrium in multicomponent systems or in condensates that were driven out of equilibrium by changes in external parameters such as temperature. In this study, we demonstrate that the aging of initially homogeneous protein-RNA condensates can spontaneously lead to the formation of kinetically arrested double-emulsion and core-shell structures without changes in external variables such as temperature or solution conditions. By combining time-resolved fluorescence-based experimental techniques with simulations based on the Cahn-Hilliard theory, we show that, as the protein-RNA condensates age, the decrease of the relative strength of protein-RNA interactions induces the release of RNA molecules from the dense phase. In condensates exceeding a critical size, aging…

ABSTRACT Intrinsically disordered proteins (IDPs) often contain proline residues, which undergo cis/trans isomerisation. While molecular dynamics (MD) simulations have the potential to fully characterise the proline cis and trans sub-ensembles, they are limited by the slow timescales of isomerisation and force field inaccuracies. Nuclear magnetic resonance (NMR) spectroscopy can report on ensemble-averaged observables for both the cis and trans proline states, but a full atomistic characterisation of these sub-ensembles is challenging. Given the importance of proline cis/trans isomerisation for influencing the conformational sampling of disordered proteins, we employed a combination of all-atom MD simulations with enhanced sampling (metadynamics), NMR, and small-angle X-ray scattering (SAXS) to characterise the two sub-ensembles of the ORF6 C-terminal region (ORF6 CTR ) from SARS-CoV-2 corresponding to the proline-57 (P57) cis and trans states. We performed MD simulations in three distinct force fields: AMBER03ws, AMBER99SB- disp , and CHARMM36m, which are all optimised for…

In order to achieve a comprehensive understanding of protein aggregation processes, an exploration of solvation dynamics, a key yet intricate component of biological phenomena, is mandatory. In the present study, we used Fourier transform infrared spectroscopy and terahertz spectroscopy complemented by atomic force microscopy and kinetic experiments utilizing thioflavin T fluorescence to elucidate the changes in solvation dynamics during liquid-liquid phase separation and subsequent amyloid fibril formation, the latter representing a transition from liquid to solid phase separation. These processes are pivotal in the pathology of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. We focus on the ACC 1–13 K 24 -ATP protein complex, which undergoes fibril formation followed by droplet generation. Our investigation reveals the importance of hydration as a driving force in these processes, offering new insights into the molecular mechanisms at play.

Morphogens are intercellular signaling molecules providing spatial information to cells in developing tissues to coordinate cell fate decisions. The spatial information is encoded within long-ranged concentration gradients of the morphogen. Direct measurement of morphogen dynamics in a range of systems suggests that local and global diffusion coefficients can differ by orders of magnitude. Further, local diffusivity can be large, which would potentially abolish any concentration gradient rapidly. Such observations have led to alternative transport models being proposed, including transcytosis and cytonemes. Here, we show that accounting for tissue architecture combined with receptor binding is sufficient to hinder the diffusive dynamics of morphogens, leading to an order of magnitude decrease in the effective diffusion coefficient from local to global scales. In particular, we built a realistic in silico architecture of the extracellular spaces of the zebrafish brain using light and electron microscopy data. Simulations on realistic architectures demonstrate that tortuosity and…

glycine and α4β2 nicotinic receptors. These structures exhibit inherent pore asymmetry, but the relevance of this to function is unknown. Furthermore, molecular dynamics simulations performed on symmetrical homomeric channels often lead to thermal distortion whereby conformations of the resulting ensemble are also asymmetrical. When functionally annotating ion channels, researchers often rely on minimal constrictions determined via radius-profile calculations performed with computer programs, such as HOLE or CHAP, coupled with an assessment of pore hydrophobicity. However, such tools typically employ spherical probe particles, limiting their ability to accurately capture pore asymmetry. Here, we introduce an algorithm that employs ellipsoidal probe particles, enabling a more comprehensive representation of the pore geometry. Our analysis reveals that the use of nonspherical ellipsoids for pore characterization provides a more accurate and easily interpretable depiction of conductance. To quantify the implications of pore asymmetry on conductance, we systematically investigated…

The angular optical trap (AOT) is a powerful instrument for measuring the torsional and rotational properties of a biological molecule. Thus far, AOT studies of DNA torsional mechanics have been carried out using a high numerical aperture oil-immersion objective, which permits strong trapping, but inevitably introduces spherical aberrations due to the glass-aqueous interface. However, the impact of these aberrations on torque measurements is not fully understood experimentally, partly due to a lack of theoretical guidance. Here, we present a numerical platform based on the finite element method to calculate forces and torques on a trapped quartz cylinder. We have also developed a new experimental method to accurately determine the shift in the trapping position due to the spherical aberrations by using a DNA molecule as a distance ruler. We found that the calculated and measured focal shift ratios are in good agreement. We further determined how the angular trap stiffness depends on the trap height and the cylinder displacement from the trap center and found full agreement between…

formation. Also, our data indicate that the HIE-stacking His is a poor acid/base catalyst when bound to the substrate and, in agreement with the literature, may play an important stabilizing role (via hydrogen bonding) during the peroxygenase catalysis. Our study reveals the pH titration midpoint values of the pH-dependent orientation of the stacking His should be considered when modeling and interpreting LPMO reactions, whether it be for classical LPMO kinetics or in industry-oriented enzymatic cocktails, and for understanding LPMO behavior in slightly acidic natural processes such as fungal wood decay.

The activities of the transient receptor potential vanilloid 4 (TRPV4), a Ca 2+ -permeable nonselective cation channel, are controlled by its surrounding membrane lipids (e.g., cholesterol, phosphoinositides). The transmembrane region of TRPV4 contains a cholesterol recognition amino acid consensus (CRAC) motif and its inverted (CARC) motif located in the plasmalemmal cytosolic leaflet. TRPV4 localizes in caveolae, a bulb-shaped cholesterol-rich domain at the plasma membrane. Here, we visualized the spatiotemporal interactions between TRPV4 and cholesterol at the plasma membrane in living cells by dual-color single-molecule imaging using total internal reflection fluorescence microscopy. To this aim, we labeled cholesterol at the cytosolic leaflets of the plasma membrane using a cholesterol biosensor, D4H. Our single-molecule tracking analysis showed that the TRPV4 molecules colocalize with D4H-accessible cholesterol molecules mainly in the low fluidity membrane domains in which both molecules are highly clustered. Colocalization of TRPV4 and D4H-accessible cholesterol was observed…