Hereditary stomatocytosis (HSt) comprises red blood cell (RBC) disorders characterized by cup-shaped erythrocytes that share a nominal morphology yet respond oppositely to splenectomy: the procedure is often curative in overhydrated HSt (OHS) and intermediate phenotypes but can precipitate life-threatening thromboembolism in dehydrated HSt (DHS/xerocytosis). This "splenectomy paradox" persists because clinicians cannot predict which patients will benefit. A central obstacle is that RBC biomechanics is governed by several partly independent parameters-membrane shear modulus (μ), bending rigidity (kc), surface-to-volume ratio (S/V), and cytoplasmic viscosity (ηcyto)-that existing assays capture only piecemeal. Here we present an in silico dissipative particle dynamics (DPD) study that constructs a control discocyte (CTR-RBC) and three stomatocyte models (ST-RBC1-3) at fixed membrane area (A0 = 132.9 μm2) and decreasing volume (V = 109.7, 101.5, 89.8 fL), spanning the OHS-to-DHS hydration range. Lacking direct membrane-mechanics data for HSt, we anchor the models to published HSt morphometry, to mechanical measurements on healthy and simulated stomatocytic cells, and to matched brightfield imaging of cup-shaped RBCs used solely as a morphological reference. Tracing this single parameter set through five mechanically orthogonal assays, we find that interendothelial-slit (IES) traversal is geometry-dominated: overhydrated ST-RBC1 requires roughly an order of magnitude higher critical pressure than CTR-RBC, whereas dehydrated ST-RBC3 passes freely. ST-RBC3 nonetheless suppresses membrane tank-treading and raises low-shear whole-blood viscosity by ∼25% at physiological haematocrit, comparable to the hyperviscosity of Gaucher-disease blood. A funnel-obstacle chip amplifies these differences into a label-free centerline-offset signal predicted to separate all four RBC types (∼4.5σ between the extreme phenotypes). Together, these results unite single-cell mechanics, splenic filtration, and hemorheology in one framework, offer a mechanical account of the OHS-versus-DHS splenectomy paradox, and point toward a microfluidic route to pre-operative risk stratification in HSt.

Human equilibrative nucleoside transporter 3 (hENT3) is a lysosomal transporter whose activity is strictly dependent on an acidic lumenal environment. Mutations in hENT3 lead to H syndrome and other metabolic disorders, yet the structural basis for its pH-sensitive gating remains poorly defined. Using multi-replica conventional MD simulations, we provide atomistic insight into the pH-dependent activation mechanism of hENT3 in the outward-facing state. Protonated Asp219 triggers a local hydrogen-bond reorganization that recruits conserved Ser229 and the TM6 helix-capping residue Asp225 and stabilizes TM5-an intrinsically flexible helix within the ENT family. Stabilization of TM5 in turn reinforces the neighboring helices including TM1, TM6 and TM8, reduces thin-gate fluctuations, and more frequently preserves the adenosine binding pose required for translocation. On the cytosolic side, Glu447 protonation modulates the interaction between the H-syndrome-associated Arg134 and loop 2. With an alignment-independent distance correlation analysis, we identified distinct state-dependent coupling patterns under acidic and neutral pH conditions. In particular, acidic ensembles that retained a transport-competent binding pose showed the strongest agreement with prior mutagenesis data, as site-centric distance correlation scores are consistent with experimentally observed loss of adenosine transport.

The Linker of Nucleus to Cytoskeleton (LINC) complex is a central mechanical element of the nuclear envelope, transmitting cytoskeletal forces to the nucleus and coordinating nuclear mechanics. While individual LINC complex units (LCUs) are well characterized structurally, growing experimental evidence suggests that LCUs assemble into higher-order clusters whose organization may critically influence force transmission. However, the molecular drivers of LINC complex clustering and their stability in the absence of externally applied forces remain poorly understood. Here, we use all-atom molecular dynamics simulations to investigate the thermodynamic and structural determinants of LINC complex clustering within the nuclear envelope. Using SUN2-KASH2 assemblies as building blocks, we generated and analyzed ten distinct higher-order cluster configurations derived from protein-protein docking, AI-based structure prediction, and biologically motivated geometric placement. Across these models, we quantified cluster stability, relative motion, interaction energetics, interacting surface area, hydrogen bond density, and per-residue effective free energy contributions. Our results reveal that LINC clustering can be stabilized through distinct interaction modes, including electrostatic, hydrogen-bond-rich, and hydrophobic interfaces, depending on cluster geometry. Stable clusters exhibit correlated motion between LCUs, minimal translational separation, and interaction energies dominated by either van der Waals or Coulombic contributions. Importantly, clustering emerges as a thermodynamically favorable property of specific LINC architectures even in the absence of applied force. Together, these findings provide a mechanistic framework for understanding LINC complex superstructure formation and suggest that LINC clustering may serve as a tunable architectural feature for regulating nuclear mechanotransduction under physiological and pathological conditions.

The Inositol 1,4,5-trisphosphate receptor channel (IP3R) is an important calcium channel involved in calcium-induced calcium release, playing a prominent role in intracellular calcium signaling. However, accurately characterizing its gating behavior remains a challenge, particularly due to the temporal resolution of patch clamp techniques that is not large enough to detect all short-lived events. This limitation can significantly bias the inference of kinetic models describing the receptor activity. To address this issue, we focused on the quantitative analysis of IP3R2 gating behavior using patch clamp data, with particular attention to missed events. We modeled IP3R2 channel gating using Hierarchical Markov chains and used a Bayesian approach that integrates missed event correction directly into the likelihood function, enabling more accurate parameter inference and model evaluation. We show that accounting for missed events deeply clarifies the multi-modal model that emerges from model selection. In this new model, the Park and Drive modes both consist of the same 3-state Markov model, with mode-dependent kinetic parameters: the Drive mode stabilizes the closed state directly connected to the open one, whereas the Park mode stabilizes the other closed state, that is not connected to the open one. Intermediate Ca[2+] concentrations are found to strongly depress the Drive to Park transition rate, so that the IP3R2 channel undergoes frequent transitions to the Park mode only for < 50 nM or micromolar Ca[2+] concentrations. Overall, our approach provides a refined perspective on IP3R2 channel modeling and highlights the critical importance of accounting for missed events upon model selection based on single-channel recordings.

A comprehensive analysis of viral mutations is essential for understanding viral evolution, disease epidemiology, diagnosis, drug resistance, and immune escape. However, challenges remain in capturing complex mutation patterns and supporting diverse viral families with varying genome architectures. To address these challenges, we present VARIANT, an openly accessible web server for mutational analysis of RNA viral genomes and associated viral products across both single- and multi-segment virus genomes. The server takes as input a viral reference genome, a reference protein sequence, and/or multiple sequence alignment, and automatically provides full annotation of mutation types, including standard categories such as point mutations (missense, silent, and nonsense), insertions, deletions, and frameshift events in both coding and non-coding regions. In addition, VARIANT detects two biologically significant mutation patterns that are often overlooked by conventional software/packages: "row mutations", "hot mutations", and separately annotates potential programmed ribosomal frameshifting regions. The server currently contains automatic analysis of major viral pathogens, including SARS-CoV-2, HIV-1, Influenza H3N2, Ebola virus, and Chikungunya virus. It also allows users to analyze additional viruses by uploading custom genome information. Users can track VARIANT analysis progress in real time, visualize mutation distributions, and download structured results in ZIP format. VARIANT also incorporates dual graph topology analysis to classify frameshifting element structures from dot-bracket notation input. This feature enables systematic comparison of RNA secondary structure motifs across viral families by mapping structures to a comprehensive library of dual graph topologies. The web server is freely available at https://variant.up.railway.app/.

The reflectins are cationic proteins that transduce neuronal signals tuning skin color for dynamic camouflage and communication in Loliginid squid. Neuronally released acetylcholine (ACh) activates phosphorylation of the reflectins, triggering their condensation, folding and hierarchical assembly. These changes drive proportional osmotic and Gibbs-Donnan dehydration of membrane-enclosed Bragg lamellae containing these proteins in skin cells called iridocytes, resulting in the calibrated, progressive change their refractive index and spacing to finely tune the wavelength of reflected light. Reflectins B and C are enriched in ACh-responsive iridocytes, suggesting they are critical for the neuronally tuned protein phase transitions within the Bragg lamellae. Here, using pH titration as an in vitro surrogate for phosphorylation to reduce protein net charge density, we demonstrate with confocal microscopy that reflectins A1, A2, B and C individually undergo LLPS to form protein dense liquid condensates, while physiological mixtures of these proteins exhibit coordinated phase transitions to form multiphase condensates whose internal spatial organization is tuned by the proportions and charge densities of the different reflectins. Our findings demonstrate that (i) the charge densities of the different reflectins control their spatial organization within liquid condensates, (ii) the relative proportions of the different reflectins found in the ACh-responsive iridocytes increase the sensitivity of their proteins' liquid phase transitions to changes in reflectin charge density, as well as (iii) suggest a biophysical explanation for the differing spatial segregation of reflectins within the iridocytes' Bragg Lamellae.

The endoplasmic reticulum (ER) is the largest continuous membrane-bound organelle in the cell and plays a central role in the synthesis and turnover of lipids and proteins. It connects directly to the nucleus through specialized contact sites known as ER-nuclear envelope (NE) junctions. In a recent study, we showed that these junctions are sparse and highly constricted, measuring less than 20 nm in diameter and occurring at a frequency of approximately 0.1 junctions per square micrometer. However, it remains unclear whether such limited and narrow connections are sufficient to support efficient transport between the ER and NE. Here, we developed a mathematical model of ER-to-NE protein diffusion that incorporates ultrastructural parameters of ER-NE junctions, their spatial frequency, and protein diffusion coefficients within the ER lumen. To validate the model, we experimentally quantified the mobility of ER luminal proteins to the NE using fluorescence recovery after photobleaching (FRAP). Both the model and experimental measurements demonstrate that passive diffusion is sufficient to account for rapid protein exchange between the ER and NE despite the sparse and constricted nature of ER-NE junctions. Furthermore, the model predicts how junction geometry and abundance influence transport efficiency. Together, these findings provide a quantitative framework for understanding ER-NE connectivity and establish that simple diffusion can account for efficient protein transport between the ER and nucleus.

In vitro reconstituted virus-like particles (VLPs) - particles with structure identical to those of virus particles except for containing non-infectious genetic information instead of a viral genome - are promising nanocarriers for delivery of therapeutic cargos. Among the simplest examples are the VLPs formed from the capsid protein (CP) of either Cowpea Chlorotic Mottle Virus (CCMV) or Brome Mosaic Virus (BMV), which form spontaneously from purified components upon mixing CCMV or BMV CP with single-stranded RNA under the right pH and ionic strength conditions. The structure of these VLPs is identical to that of the virus particles, consisting of 180 copies of the CP organized as 12 icosahedrally-symmetric pentamers separated by 20 hexamers. In contrast, most viruses that infect mammalian cells are enveloped by a lipid bilayer filled with transmembrane virally-encoded glycoproteins, and it has not yet proved possible to reconstitute these particles from their purified components. To take advantage of the robustness of unenveloped virus capsids and the biocompatibility of lipid-bilayer-wrapped enveloped viruses, we demonstrate in vitro lipid-bilayer wrapping of intact plant viral (CCMV and BMV) capsids. Lipid compositions are chosen to match the opposite charge of the viral capsid, to promote stable membrane association. Our results provide an important first step toward the wrapping of in vitro reconstituted VLPs containing therapeutic mRNA. These enveloped virus-like particles (eVLPs) combine the stability and scalability of plant virus capsids with the functional advantages of lipid envelopes, advancing their potential as platforms for RNA delivery, vaccine design, and therapeutic nanotechnology.

(≤300 words, current = 296): Retinal cone photoreceptor outer segments (COS) are modified cilia where light is transduced into electrical signals. While extensive ultrastructural studies have established that the COS membrane system consists of several hundred parallel lamellae interconnected by the ciliary plasma membrane (PM) through narrow membrane bridges known as saddle points (SP), little is known about how components of the phototransduction cascade are delivered to COS, how they are loaded into flat lamellae surfaces (LS) and how they maintain uniform distribution among lamellae, especially in light of the steeply tapered lamellar geometry found in some species and high membrane turnover rate. Here, we quantify interlamellar membrane protein flux in Xenopus COS using a neutral membrane probe - the transmembrane domain of syntaxin 3 fused with EGFP (EGFP-Stx3TM), and develop 3D and 1D computational models of protein transport within the COS membrane system. The models describe diffusional transport of the probes along the folded membranes accounting for effects of molecular crowding. Axial equilibration of EGFP-Stx3TM in FRAP experiments is quantitatively described by the model based on diffusional transport, alone. The diffusion coefficient on the PM obtained from the model fitted to experimental data is approximately equal to that on the LS. Sensitivity analysis of the model shows that, surprisingly, SP contribute only ∼15% control of axial transport while the chief control, ∼80%, is located at the PM, and this control distribution is robust to changes in conditions. Thus, COS plasma membrane acts as a shunt conducting free transmembrane proteins between lamellae by Brownian diffusion, even in the presence of crowding effects and complex membrane geometry, while lamellae are capacitive sources/sinks. Axial Stx3TM flux can exceed 100 molecules/s suggesting that if opsins diffuse freely, their flux may exceed 104 molecules/s, supporting rapid equilibration and uniform distribution throughout the contiguous COS membrane system.

Rhythmic contraction of cardiomyocytes is driven by self-sustained intracellular calcium oscillations. While these oscillations are typically attributed to intrinsic calcium cycling, increasing experimental evidence indicates that external mechanical forces can modulate calcium oscillation frequency and lead to frequency entrainment. However, the physical mechanisms underlying such mechanical entrainment remain unclear. Here, we develop a minimal mechanochemical model that couples intracellular calcium cycling, adaptive ryanodine receptor (RyR) gating, and strain-induced reactive oxygen species (ROS) signaling to investigate how external mechanical forces reshape the nonlinear dynamics of autonomous calcium oscillations in cardiomyocytes. Linear stability analysis reveals that RyR inactivation is a slow adaptive variable that sets the intrinsic calcium oscillation timescale and stabilizes limit-cycle dynamics. Within this framework, external mechanical forces act as a parametric perturbation that reshapes the phase-space structure of the oscillator. We show that periodic forces induce frequency entrainment within a finite locking range, followed by a transition to a bursting-like regime as the force frequency exceeds a critical threshold. This transition boundary is further tunable by the balance between ROS production and clearance, which controls the effective sensitivity of the oscillator. These results thus establish a quantitative dynamical framework linking RyR channel kinetics to the stability and synchronization of cardiac calcium oscillations, and provide mechanistic insight into frequency-dependent mechanochemical coupling in cardiomyocytes.

A broad range of cellular functions involve transient or persistent changes in the morphology of lipid membranes, from the organellar to the molecular scale. By and large, the thermodynamics of protein-mediated remodeling, which often distort the membrane beyond its thermally accessible undulations, remain to be understood. Molecular Dynamics simulations enhanced by advanced sampling methods are uniquely suited to examine and quantitate these phenomena. Here, we leverage the Multi-Map simulation method to directly quantify how applied lateral tension impacts the energetics of both global and localized membrane perturbations induced extrinsically. We obtain free-energy profiles of curvature and thickness deformations with and without tension, which allow us to test the quadratic approximations of Helfrich-based continuum theories. We find that these continuum models are applicable up to ∼ 20 kBT, beyond which they under-estimate the free-energy cost of bending. However, we find that the effect of tension on these deformations is primarily driven by changes in the projected area of the membrane, and therefore consistent with continuum theory, even beyond 30 kBT. These changes outpace the effects of tension on lipid packing and diffusion, and importantly we find that tension levels preceding rupture (≤ 10 mN/m) do not impede complete lipid mixing. Finally, we investigate the free-energy landscapes of membrane thickness and lipid enrichment with a heterogeneous lipid composition and show that strong enrichment can be sustained even under steady exchange of the constituent lipid molecules.

Staphylococcus aureus presents significant challenges in clinical settings due to its ability to form resilient biofilms, which drive persistent infections on medical implants and at wound sites. These bacteria are the leading cause of biomaterial-associated infections, often leading to implant failure. Moreover, S. aureus can form biofilms on cardiovascular implants, further complicating device-related infections. Cavitation microstreaming, generated by ultrasound-activated microbubbles through their oscillations, has emerged as a promising method for disrupting biofilms. However, the underlying biofilm-microstreaming interactions governing bacterial detachment remain poorly understood. In this study, a computational framework integrating coarse-grained molecular dynamics with the lattice Boltzmann method was developed to simulate Staphylococcus aureus biofilm growth and its response to microstreaming. The modeled biofilm morphology closely aligns with experimental confocal microscopy data, with less than 14% deviation in spatial spreading and roughness. To assess biofilm disruption, we applied microstreaming fields with dipole and quadrupole flow patterns at representative speeds of 1, 5, and 10 mm/s. Our results demonstrate that higher flow velocities facilitate greater detachment, with dipole flows inducing gradual erosion and quadrupole flows causing more localized disruption. By integrating computational modeling with experimental validation, this work provides a robust platform to investigate and maximize biofilm-microstreaming interactions. These findings emphasize the potential of ultrasound-activated microbubble-induced microstreaming as a noninvasive strategy for biofilm removal and provide a foundation for future therapeutic approaches.

Members of kinesin-14 family motors, which can move toward the microtubule (MT) minus end, play roles in spindle assembly and maintenance during mitosis and meiosis. Some kinesin-14 motors are non-processive while others are processive. Here, we study both theoretically and numerically the dynamics of the MT gliding and relative sliding of antiparallel MTs by multiple kinesin-14 motors. We show that while the published experimental data on the MT gliding and sliding by multiple kinesin-14 HSET motors cannot be explained by considering that the HSET is non-processive, they are well explained by a model in which HSET has a small processivity. More interestingly, we find that an optimal small processivity of the kinesin-14 motor is present, under which the MT sliding by the multiple motors can attain maximal velocity. Moreover, we show that the non-processive motors have an evidently larger ability to accumulate at the minus ends of parallel MTs than the processive motors of even a very small processivity. These imply that while the kinesin-14 motors of the small processivity are more likely to function as maintaining antiparallel MTs in spindles, the non-processive motors are more likely to function as anchoring nearly parallel MTs at their minus ends to form spindle poles.

A common explanation for brightness variation in fluorescent proteins (FPs) is that tight chromophore cages produce bright proteins. This model predicts that quantum yield should track the dihedral rotational space available to the chromophore. We tested that prediction across 838 FP crystal structures from the Protein Data Bank. For each structure, we scanned the chromophore's two methine-bridge torsions, τ and φ. We measured two features of the chromophore environment: the fraction of torsional space that is sterically accessible, and the angular distance from the deposited chromophore to the nearest planar geometry. The scan shows a consistent family-wide asymmetry. The I-bond, the cis-trans isomerization axis, is clamped in all color classes. The P-bond, the ring-rotation axis, is the variable degree of freedom. Gatekeeper analysis identifies position 203, in avGFP numbering, as the dominant P-bond constraint. It is the first steric barrier in 21% of all sweep events. Cage size does not generally track quantum yield; instead, it primarily reflects chromophore chemistry. Indole-based cyan FPs and acylimine-based red FPs have the tightest cages, while imidazole-based blue FPs have the loosest. In red FPs, quantum yield is associated instead with ground-state chromophore geometry. More twisted crystallographic chromophores are consistently dimmer in red FPs. The scan also shows that rigidity is not a single property. Cage size, ground-state geometry, gatekeeper residues, and thermal immobilization are distinct measures of rigidity, each with a different relationship to quantum yield. Fluorescent-protein design should therefore target local control of chromophore geometry rather than global cage tightening.