Biophysical journal最新文献

Biological condensates often emerge as a multidroplet 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 multidroplet 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 intercondensate 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 dynamic nature of protein-RNA condensates.

The mechanically-activated ion channel PIEZO1 is critical to numerous physiological processes, and is activated by diverse mechanical cues. The channel is gated by membrane tension and has been found to be mobile in the plasma membrane. We employed single particle tracking (SPT) of endogenous, tdTomato-tagged PIEZO1 using Total Internal Reflection Fluorescence Microscopy in live cells. Application of SPT unveiled a surprising heterogeneity of diffusing PIEZO1 subpopulations, which we labeled "mobile" and "immobile". We sorted these trajectories into the two aforementioned categories using trajectory spread. To evaluate the effects of the plasma membrane composition on PIEZO1 diffusion, we manipulated membrane composition by depleting or supplementing cholesterol, or by adding margaric acid to stiffen the membrane. To examine effects of channel activation on PIEZO1 mobility, we treated cells with Yoda1, a PIEZO1 agonist, and GsMTx-4, a channel inhibitor. We collected thousands of trajectories for each condition, and found that cholesterol removal and Yoda1 incubation increased the channel's propensity for mobility. Conversely, we found that GsMTx-4 incubation and cholesterol supplementation resulted in a lower chance of mobile trajectories, whereas margaric acid incubation did not have a significant effect on PIEZO1 mobility. The "mobile" trajectories were analyzed further by fitting the time-averaged mean-squared displacement as a function of lag time to a power-law model, revealing mobile PIEZO1 puncta exhibit anomalous subdiffusion. These studies illuminate the fundamental properties governing PIEZO1 diffusion in the plasma membrane and set the stage to determine how cellular processes and interactions may influence channel activity and mobility.

Fluorine-19 is an ideal nucleus for studying biological systems using NMR due to its rarity in biological environments and its favorable magnetic properties. In this work, we used a mixture of monofluorinated palmitic acids (PAs) as tracers to investigate the molecular interaction of the fluorinated drug rosuvastatin in model lipid membranes. More specifically, PAs labeled at the fourth and eighth carbon positions of their acyl chains were coincorporated in phospholipid bilayers to probe different depths of the hydrophobic core. First, the ¹⁹F chemical shift anisotropy (CSA), indicative of membrane fluidity, was simultaneously determined for fatty acids (FAs) and the fluorinated drug using either slow magic-angle spinning (MAS) 1D ¹⁹F solid-state NMR (SS-NMR) or MAS 2D ¹⁹F-¹⁹F SS-NMR with CSA recoupling. Membrane heterogeneity and selective partitioning of rosuvastatin into fluid regions could thus be evidenced. We then examined the possibility of mapping intermolecular distances in bilayers, in both the fluid and gel phases, using ¹⁹F-¹⁹F and ¹H-¹⁹F correlation experiments by SS-NMR using MAS. Spatial correlations were evidenced between the two PAs in the gel phase, while contacts between the statin and the lipids were detected in the fluid phase. This work paves the way to mapping membrane-active molecules in intact membranes, and stresses the need for new labeling strategies for this purpose.

The SLC (solute carrier) superfamily mediates the passive transport of small molecules across apical and basolateral cell membranes in nearly all tissues. In this paper, we employ bond-graph approaches to develop models of SLC transporters that conserve mass, charge, and energy, respectively, and can be parameterized for a specific cell and tissue type for which the experimental kinetic data are available. We show how analytic expressions that preserve thermodynamic consistency can be derived for a representative four- or six-state model, given reasonable assumptions associated with steady-state flux conditions. We present details on fitting parameters for SLC2A2 (a GLUT transporter) and SLC5A1 (an SGLT transporter) to experimental data and show how well the steady-state flux expressions match the full kinetic analysis. Since the bond-graph approach will not be familiar to many readers, we provide a detailed description of the approach and illustrate its application to a number of familiar biophysical processes.

The accumulation-associated protein (Aap) is the primary determinant of Staphylococcus epidermidis device-related infections. The B-repeat superdomain is responsible for intercellular adhesion that leads to the development of biofilms occurring in such infections. It was recently demonstrated that Zn-induced B-repeat assembly leads to formation of functional amyloid fibrils, which offer strength and stability to the biofilm. Rigorous biophysical studies of Aap B-repeats from S. epidermidis strain RP62A revealed Zn-induced assembly into stable, reversible dimers and tetramers, prior to aggregation into amyloid fibrils. Genetic manipulation is not tractable for many S. epidermidis strains, including RP62A; instead, many genetic studies have used strain 1457. Therefore, to better connect findings from biophysical and structural studies of B-repeats to in vivo studies, the B-repeat superdomain from strain 1457 was examined. Differences between the B-repeats from strains RP62A and 1457 include the number of B-repeats, which has been shown to play a critical role in assembly into amyloid fibrils, as well as the distribution of consensus and variant B-repeat subtypes, which differ in assembly competency and thermal stability. Detailed investigation of the Zn-induced assembly of the full B-repeat superdomain from strain 1457 was conducted using analytical ultracentrifugation. Whereas the previous construct from RP62A (Brpt5.5) formed a stable tetramer prior to aggregation, Brpt6.5 from 1457 forms extremely large stable species on the order of ≈28-mers, prior to aggregation into similar amyloid fibrils. Our data suggest that both assembly pathways may proceed through the same mechanism of dimerization and tetramerization, and both conclude with the formation of amyloid-like fibrils. Discussion of assembly behavior of B-repeats from different strains and of different length is provided with considerations of biological implications.

Stomatocyte-discocyte-echinocyte (SDE) transformations in human red blood cells (RBCs) have significant influences on blood dynamics and related disorders. The mechanical properties of the RBC membrane, such as shear modulus and bending elasticity, play crucial roles in determining RBC shapes. Recent biophysical findings reveal that building a comprehensive model capable of describing SDE shape transformations is a challenging problem. Based on dissipative particle dynamics, this study develops a two-component RBC model considering the detachment between the lipid bilayer and cytoskeleton, as well as the cytoskeletal reorganization during echinocyte formation. This model is validated by comparing RBCs' geometric shape and the apparent membrane tension with previous experimental measurements. Results indicate that a complete SDE sequence represented by six typical shapes can be obtained by modulating the model's mechanical and geometric parameters. Furthermore, a phase diagram based on reduced variables is obtained using principal-component analysis, demonstrating the phase transformations among SDE shapes. Our result suggests that the transformation from discocyte to stomatocyte is primarily influenced by dimensionless bending rigidity, whereas, during echinocyte formation, three key variables, i.e., dimensionless bending rigidity, targeting cytoskeleton shrinkage ratio, and connecting pattern, have joint impacts on the formation of spicules or bumps and the development of the cytoskeletal framework. The present two-component RBC model and the associated findings provide a perspective for a deeper understanding of the SDE transformation mechanism. This framework offers new insights into biological science and potential applications in the field of biomedical engineering.

Breast tumors are typically surrounded by extracellular matrix (ECM), which is heterogeneous, not just structurally but also mechanically. Conventional rheometry is inadequate for describing cell-size-level spatial differences in ECM mechanics that are evident at micrometer scales. Optical tweezers and passive microrheometry provide a microscale resolution for the purpose but are incapable of measuring ECM viscoelasticity (the liquid-like viscous and solid-like elastic characteristics) at stiffness levels as found in breast tumor biopsies. Magnetic microrheometry records data on varying microscale viscoelasticity within 3D ECM-mimicking materials up to the biopsy-relevant stiffness. However, the measurement probe-based microrheometry data has limitations in spatial resolution. Here, we present a probabilistic modeling method-providing analysis of sparse, probe-based spatial information on microscale viscoelasticity in ECM obtained from magnetic microrheometry-in two parts. First, we validate the method's applicability for analysis of a controlled stiffness difference, based on two collagen type 1 concentrations in one sample, showing a detectable stiffness gradient in the interface of the changing concentrations. Second, we used the method to quantify and visualize differences in viscoelasticity within 3D cell cultures containing breast-cancer-associated fibroblasts, and collagen type 1 (both typically present in the tumor ECM). The fibroblasts' presence stiffens the collagen material, which aligns with previous research. Importantly, we provide probabilistic quantification of related spatial heterogeneity differences in viscoelasticity recorded by magnetic microrheometry, for the first time. The fibroblasts culturing leads to an initially higher spatial heterogeneity in the collagen stiffness. In summary, this method reports on enhanced spatial mapping of viscoelasticity in breast cancer 3D cultures, with the future potential for matching of spatial viscoelasticity distribution in the 3D cultures with the one in biopsies.

Ca²⁺ blinks measure the exit of Ca²⁺ from the junctional sarcoplasmic reticulum (JSR) in a cardiac myocyte during a Ca²⁺ spark. Here, the relationship between experimental blink fluorescence measurements and the [Ca²⁺] in the JSR is explored using long 3D simulations of diastolic Ca²⁺ release. For a fast intra-SR Ca²⁺-activated fluorophore such as Fluo-5N, we show that a simple mathematical formula relates the two for an ideal blink (i.e., when fluorescence signals come only from the JSR). The formula shows that normalized JSR [Ca²⁺] is much lower than the normalized fluorescence and that JSR Ca²⁺ depletes ∼40-50% more than previously inferred from blink fluorescence measurements. In addition, we show that stray fluorescence signals (e.g., from other parts of the sarcoplasmic reticulum network) can mask even deeper Ca²⁺ depletion. Overall, the simulations show that strong JSR Ca²⁺ depletion such as that seen in many simulations is consistent with the relatively moderate fluorescence changes seen in experiments.

Experimentally monitoring the kinematics of branching network growth is a tricky task, given the complexity of the structures generated in three dimensions. One option is to drive the network in such a way as to obtain two-dimensional growth, enabling a collection of independent images to be obtained. The density of the network generates ambiguous structures, such as overlaps and meetings, which hinder the reconstruction of the chronology of connections. In this paper, we propose a general method for global network reconstruction. Each network connection is defined by a unique label, enabling it to be tracked in time and space. In this work, we distinguish between lateral and apical branches on the one hand, and extremities on the other. Finally, we reconstruct the network after identifying and eliminating overlaps. This method is then applied to the model filamentous fungus Podospora anserina to reconstruct its growing thallus. We derive criteria for differentiating between apical and lateral branches. We find that the outer ring is favorably composed of apical branches, while densification within the network comes from lateral branches. From this, we derive the specific dynamics of each of the two types. Finally, in the absence of any latency phase during growth initiation, we can reconstruct a time based on the equality of apical and lateral branching collections. This makes it possible to directly compare the growth dynamics of different thalli.

During the active transport of cellular cargo, forces generated by cargo-associated molecular motors propel the cargo along cytoskeletal tracks. However, the forces impact not only the cargo, but also the underlying cytoskeletal filaments. To better understand the interplay between cargo transport and the organization of cytoskeletal filaments, we employ coarse-grained computer simulations to study actin filaments interacting with cargo-anchored myosin motors in a confined domain. We show that cargo transport can lead to the segregation of filaments into domains of preferred filament polarity separated by clusters of aggregated cargoes. The formation of polarity-sorted filament domains is enhanced by larger numbers of cargoes, more motors per cargo, and longer filaments. Analysis of individual trajectories reveals dynamic and heterogeneous behavior, including locally stable aggregates of cargoes that undergo rapid coalescence into larger clusters when sufficiently close. Our results provide insight into the impact of motor-driven organelle transport on actin filaments, which is relevant both in cells and in synthetic environments.