Soft Matter

The remarkable elastic properties of polymers are ultimately due to their molecular structure, but the relation between the macroscopic and molecular properties is often difficult to establish, in particular for (bio)polymers that contain hydrogen bonds, which can easily rearrange upon mechanical deformation. Here we show that two-dimensional infrared spectroscopy on polymer films in a miniature stress tester sheds new light on how the hydrogen-bond structure of a polymer is related to its visco-elastic response. We study thermoplastic polyurethane, a block copolymer consisting of hard segments of hydrogen-bonded urethane groups embedded in a soft matrix of polyether chains. The conventional infrared spectrum shows that upon deformation, the number of hydrogen bonds increases, a process that is largely reversible. However, the 2DIR spectrum reveals that the distribution hydrogen-bond strengths becomes slightly narrower after a deformation cycle, due to the disruption of weak hydrogen bonds, an effect that could explain the strain-cycle induced softening (Mullins effect) of polyurethane. These results show how rheo-2DIR spectroscopy can bridge the gap between the molecular structure and the macroscopic elastic properties of (bio)polymers.

Cell plasma membranes exhibit heterogeneous lateral organization whose dynamic compartmentalization is critical for processes such as viral infection and fertilization. While membrane tension is known to influence crucial cell remodeling processes, its role in regulating membrane heterogeneous organization remains unclear. To reveal the effect of tension on lateral membrane organization, we used supported lipid bilayers on flexible substrates. These were prepared by rupturing ternary-composition giant unilamellar vesicles exhibiting liquid order-disorder phase coexistence. The phase coexistence is observed using a fluorescent probe that preferentially partitions to the disordered phase. Using a motorized equibiaxial stretching device, we observed domain morphology homogenization under membrane stretching. We define an order parameter based on the relative concentration of the dye in the two phases, which is a proxy for the membrane lateral organization. Order parameter analysis revealed power-law scaling near the critical strain with an exponent {beta} = 1.0 {+/-} 0.3, consistent with an elastic theoretical model predicting {beta} = 1. The progressive broadening of the interfacial region width near the critical strain, and continuous transition to a homogeneous phase, is consistent with a second-order phase transition. These findings indicate that membrane tension may serve as a physical regulator of lateral lipid organization, with implications for how cells use mechanical forces to regulate their structure and function.

The spontaneous directional motion of vesicles on both the outer and inner surfaces of a conical substrate is observed in this work. We showed that the motion is ultra-fast and the maximum velocity can be as high as 2.14 nm/s. The driving force behind is attributed to the reduction of the bending energy along the conical surface, which possesses high curvature gradient. SummaryWe observed and explained the spontaneous directional motion of vesicles on both the concave and convex surfaces of a cone.

The primary event in chemical neurotransmission involves the fusion of a membrane-limited vesicle at the plasma membrane and the subsequent release of its chemical neurotransmitter cargo. The cargo itself is not known to have any effect on the fusion event. However, amphiphilic monoamine neurotransmitters (e.g. serotonin and dopamine) are known to strongly interact with lipid bilayers and to affect their mechanical properties, which can in principle impact membrane-mediated processes. Here we probe whether serotonin can enhance the association and fusion of artificial lipid vesicles in vitro. We employ Fluorescence Correlation Spectroscopy and Total Internal Reflection Fluorescence microscopy to measure the attachment and fusion of vesicles whose lipid compositions mimic the major lipid components of synaptic vesicles. We find that association between vesicles and supported lipid bilayers are strongly enhanced in a serotonin dose-dependent manner, and this drives an increase in the rate of spontaneous fusion. Molecular dynamics simulations and fluorescence spectroscopy data show that serotonin insertion increases the water content of the hydrophobic part of the bilayer. This suggests that the enhanced membrane association is likely driven by an energetically favourable drying transition. Other monoamines such as dopamine and norepinephrine, but not other related species such as tryptophan, show similar effects on membrane association. Our results reveal a lipid bilayer-mediated mechanism by which monoamines can themselves modulate vesicle fusion, potentially adding to the control toolbox for the tightly regulated process of neurotransmission in vivo. TOC graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/576155v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@2717c5org.highwire.dtl.DTLVardef@899c21org.highwire.dtl.DTLVardef@697232org.highwire.dtl.DTLVardef@c850a0_HPS_FORMAT_FIGEXP M_FIG C_FIG

The creation of free-standing lipid membranes has been so far of remarkable interest to investigate processes occurring in the cell membrane since its unsupported part enables studies in which it is important to maintain cell-like physicochemical properties of the lipid bilayer, that nonetheless depend on its molecular composition. In this study, we prepare pore-spanning membranes that mimic the composition of plasma membranes and perform force spectroscopy indentation measurements to unravel mechanistic insights depending on lipid composition. We show that this approach is highly effective for studying the mechanical properties of such membranes. Furthermore, we identify a direct influence of cholesterol and sphingomyelin on the elasticity of the bilayer and adhesion between the two leaflets. Eventually, we explore the possibilities of imaging in the unsupported membrane regions. For this purpose, we investigate the adsorption and movement of a peripheral protein, the fibroblast growth factor 2, on the complex membrane.

Statistical mechanics is employed to tackle the problem of polymer brush bilayers under stationary shear motion. The article addresses, solely, the linear response regime in which the polymer brush bilayers behave very much similar to the Newtonian fluids. My approach to this long-standing problem split drastically from the work already published Kreer, T., Soft Matter, 12, 3479 (2016). It has been thought for many decades that the interpenetration between the brushes is source of the friction between the brush covered surfaces sliding over each other. Whiles, in the present article I strongly reject that idea. Instead, here, I show that structure of the whole system is responsible for friction between brush covered surfaces and the interpenetration is absolutely insignificant. Two simple reasons for that are the presence of ambient solvent and also flexibility of the chains. The results of this research would blow ones mind about how the polymer brush bilayers respond at small shear rates.

Examining the design principles of biological materials, in particular the presence of inhomogeneities in their ultrastructure is the key to understanding the often remarkable mechanical properties possessed by them. In this work, motivated by the question of understanding the effect of variability in the material properties of the peptide cross-linkers on the bulk mechanical properties of the cell wall structure of bacteria, we study a spring system in which variability is encoded by assigning values of spring constants and rupture strengths of the constituent springs from appropriate probability distribution. Using analytical methods and computer simulations, we study the response of the spring system to shear loading and observe how heterogeneities inherent in the system can heighten the resistance to failure. We derive the force extension relation of the system and explore the effect that the disorder in values of spring constant and rupture strength has on load carrying capacity of the system and failure displacement. We also study a discrete step shear loading of the system, exhibiting a transition from quasi-brittle to brittle response controlled by the step size, providing possible framework to experimentally quantify the disorder in analogous structures. The model studied here will also be useful in general to understand fiber bundles exhibiting disorder in the elasticity and rupture strengths of constituent fibers.

Biomolecular condensates have so far been studied in terms of their structural, compositional, and functional properties. However, condensate enzymatic activity --a key aspect of cellular metabolism-- remains unexplored due to the complexity of the system. In this study, using a combination of experimental, computational and theoretical techniques, we have discovered that the non-equilibrium activity which originates from catalytic reactions couples with the environment through various feedback mechanisms across five orders of magnitude of length scales. We observe that condensed enzymes catalyse more rapidly in the presence of crowding proteins and show that the increased enzymatic activity within these droplets stems from the emergence of lower-energy protein conformations induced by the highly crowded environment. Despite the crowding in the environment of the droplet, which might suggest an effective increase in its overall viscosity, we find that it becomes more agile, as evidenced by the observation of enhanced diffusion and macroscopic flow, due to the enzymatic activity. These findings shed new light on the dynamic interplay between enzymatic activity, composition and crowding in condensates, and their roles on the mobility and accessibility of various functional units in these environments, offering a novel perspective on liquidliquid phase separation in metabolically active conditions.

Many internal organs in multicellular organisms comprise epithelia which enclose fluid-filled cavities. These are referred to as lumens and their formation is regulated by a wide range of processes, including epithelial polarization, secretion, exocytosis and actomyosin contractility [1, 2]. While these mechanisms have shed light on lumen growth, what controls lumen morphology remains enigmatic. Here we use pancreas organoids to explore how lumens acquire either a spherical shape or a branched topology [3]. Combining computational simulations based on a phase field model with experimental measurements we reveal that lumen morphology arises from the balance between the cell cycle duration and lumen pressure, with more complex lumen at low pressure and fast proliferation rates. Moreover, the manipulation of proliferation and lumen pressure in silico and in vitro is sufficient to alter and reverse the morphological trajectories of the lumens. Increasing epithelial permeability of spherical lumens lead to lower lumen pressure and converts their morphology to complex lumen shapes, highlighting its crucial role. In summary, the study underscores the importance of balancing cell proliferation, lumen pressure, and epithelial permeability in determining lumen morphology, providing insights relevant to other organs, for tissue engineering and cystic disease understanding and treatment [4].

We study the role of a biomimetic actin cortex during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model system. The actin cortex, a subjacently attached interconnected network of actin filaments, regulates the shape and mechanical properties of the plasma membrane of mammalian cells, and is a major factor influencing the mechanical response of the cell to external physical cues. We demonstrate that the presence of an actin shell inhibits the formation of macropores in the electroporated GUVs. Additionally, experiments on the uptake of dye molecules after electroporation show that the actin network slows down the resealing process of the permeabilized membrane. We further analyze the stability of the actin network inside the GUVs exposed to high electric pulses. We find disruption of the actin layer that is likely due to the electrophoretic forces acting on the actin filaments during the permeabilization of the GUVs. Our findings on the GUVs containing a biomimetic cortex provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV.

The spatial organization of membrane-associated proteins is essential for a wide range of cellular processes, including signal transduction, endocytosis, and cell adhesion. While protein clustering can be driven by direct short-range forces, indirect interactions mediated by the membrane itself, particularly those arising from thermal shape fluctuations, are potentially sufficient to drive clustering in the absence of direct binding. In this study, we investigate the capacity of fluctuation-induced interactions to induce clustering using mesoscale simulations based on dynamically triangulated surfaces. We examine the roles of protein-induced changes in the local bending rigidity and Gaussian modulus, as well as the effects of protein concentration, identifying a crossover between dispersed and aggregated states that depends on these three parameters. We further explore the influence of surface tension, finding that tension mildly reduces clustering far from the crossover point but has a pronounced effect near it. We generalize these observations to spherical geometries, reporting similar results relevant to experiments involving small unilamellar vesicles (SUVs). Extending the model to systems with two types of stiff inclusions, we show that stiff proteins can serve as clustering centers for less stiff proteins. Finally, we analyze the impact of protein-induced preferred membrane curvature. In this scenario, the combination of fluctuation-mediated clustering and curvature induction can drive membrane shape remodeling. The non-additive nature of fluctuation-induced forces poses a challenge to predicting collective behavior, but our simulations provide a comprehensive framework that unifies previous observations. These findings highlight how a few mesoscale physical parameters can control protein self-organization on membranes, offering insights relevant to both cell biology and the design of membrane-associated nanoparticles.

Adhesion domains forming at the membrane interfaces between two cells or a cell and the ex-tracellular matrix commonly involve multiple proteins bridges. However, the physical mechanisms governing the domain structures are not yet fully resolved. Here we present a joint experimental and theoretical study of a mimetic model-system, based on giant unilammelar vesicles interacting with supported lipid bilayers, with which the underlying physical effects can be clearly identified. In our case, adhesion is induced by simultaneous action of DNA linkers with two different lengths. We study the organization of bridges into domains as a function of relative fraction of long and short DNA constructs. Irrespective of the composition, we systematically find adhesion domains with coexisting DNA bridge types, despite their relative differences in length of 9 nm. However, at short length scales, below the optical resolution of the microscope, simulations suggest the formation of nanodomains by the minority fraction. The nano-aggregation is more significant for long bridges, which are also more stable, even though the enthalpy of membrane insertion is the same for both species.

Motivated by the work on block copolymer models that provide insights into epigenetics driven chromosome organization, we investigate the segregation behavior of five distinct 2-block co-polymers (BCPs) system with varying block sizes, confined within both symmetric and lateral geometries. Using exact enumeration method and Langevin dynamics simulation, our simple self-avoiding polymer model reveals robust behaviors (across statics and dynamic studies) despite strong finite-size effects. We observe that as block length increases, polymer compaction intensifies relying on non-specific interaction, leading to longer segregation times. The dynamic study clearly demonstrates the formation of globular lamellar phases and condensed, stable complex structures in long-range block copolymer (BCP) systems, providing a simplified analogy to lamellar-mediated chromatin compaction, which involves structures that are difficult to segregate under physiological conditions. Dominance of specific interaction over non-specific interaction in long range BCP systems leads to phase separation driven self assemblies which provides a simplified analogy to heterochromatin--inactive or stable domains. In contrast, short-range block sequences remain in a coiled state, exhibiting minimal overlap or interaction due to strong short range attraction, which may corresponds to euchromatin regions where diverse epigenetic states coexist, resulting in active, non-condensed structures. We also observe that asymmetric or lateral confinement favors more segregation between the BCPs irrespective of their underlying sequence.

-synuclein is a neuronal protein implicated in neurotransmitter release. Its function is thought to critically depend on the dynamic equilibrium between free and membrane-bound protein. -synuclein amyloid formation implicated in Parkinsons Disease was also shown to be modulated by lipid membranes. However, it remains elusive whether -synuclein-related pathology is due to loss-of-function or gain-of-toxic-function. To help address this question, we studied the coupling of the equilibrium between free and membrane-bound -synuclein and membrane-induced amyloid formation - phenomena that are usually treated separately. We present a description of the system on a wide range of length scales and timescales for lipid-to-protein ratio conditions where amyloid formation is either accelerated or inhibited by lipid membranes. We find a clear difference between the dynamics and heterogeneity of the protein-covered membrane interface in the two sets of conditions. In aggregation-accelerating conditions, the membrane interface is dynamic and heterogeneous with rapid exchange between free and membrane-bound protein, and disordered protein segments of varying lengths exposed to solution. All these characteristics of the membrane interface are likely to decrease the free energy barrier for amyloid formation. Conversely, the membrane interface is homogeneous and less dynamic in conditions where amyloid formation is inhibited. Importantly, any factors affecting the equilibrium between free and membrane-bound -synuclein may trigger a change from non-aggregating to aggregating conditions. Altogether, our results highlight a strong coupling of the dynamic equilibrium between the free and membrane-bound -synuclein and membrane-modulated amyloid formation and thus of the physiological function of -synuclein and its aberrant aggregation.

Elastic properties of nanoscale extracellular vesicles (EVs) are believed to influence their cellular interactions, thus having a profound implication in intercellular communication. Yet, an accurate quantification of the elasticity of such small lipid vesicles is difficult even with AFM-based nanoindentation experiments as it crucially depends on the reliability of the theoretical interpretation of such measurements. Here we describe a complete method composed of theoretical framework, experimental procedure, and appropriate statistical approach for an accurate determination of bending modulus and effective elastic modulus of EVs. Further, we experimentally demonstrate that the quantification of EVs by the elastic modulus from AFM-based force spectroscopy measurement is marred by the interplay of their compositionally inhomogeneous fluid membrane with the adhesion forces from the substrate and thermal effects - two exquisite phenomena that could thus far only be theoretically predicted. The effects result in a large spreading of elastic modulus even for a single EV. Our unified model is then applied to genetically engineered classes of EVs to understand how the alterations in tetraspanin expression may influence their elastic modulus.

FET (FUS-EWSR1-TAF15) family proteins inherently form mesoscale molecular assemblies, known as clusters, under physiological conditions at concentrations well below the threshold for phase separation. This study demonstrates that adenosine triphosphate (ATP), an amphiphilic molecule and essential cellular metabolite, modulates the size of these sub-saturation mesoscale clusters in a concentration-dependent manner. At low concentrations (1-2 mM), ATP acts as a crosslinker for FET proteins, resulting in larger size clusters. At moderate concentrations (5 mM), the size of the clusters decreases but stabilizes. At high concentrations (10 mM), the cluster size further diminishes. Other amphiphilic molecules, including common hydrotropes like sodium xylene sulfonate, sodium toluene sulfonate, and hexanediol, exhibit comparable concentration-dependent effects on FET protein clustering. Notably, these effects cannot be explained solely by hydrotropic or kosmotropic mechanisms; instead, they stem from non-specific interactions between proteins and small molecules. The intrinsic chemical properties of the amphiphilic molecules and FET proteins play a crucial role in regulating mesoscale cluster formation at sub-saturation concentrations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/649119v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@90de68org.highwire.dtl.DTLVardef@f9bcb5org.highwire.dtl.DTLVardef@1d42e59org.highwire.dtl.DTLVardef@12eed97_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mucus is a fluid that protects animals against pathogens while promoting interactions with commensal microbes. Changes in the diffusivity of particles in mucus alter viruses infectivity, the efficiency of bacterial pathogens to invade a host, and the effectivity of drug delivery. Multiple physicochemical properties modulate the diffusion of microscopic particles in mucus, but their combined effect is unclear. Here, we analyzed the impact of particle size, charge, chemistry, anomalous diffusion exponent, and mucus composition in the diffusivity of particles from 106 published experiments. We used a time window sampling of one second to define a consistent, effective diffusion across experiments. The effective diffusion spanned seven orders of magnitude from 10-5 to 102 {micro}m2/s. The anomalous exponent was the strongest predictor among all variables tested. It displayed an exponential relationship with the effective diffusion that explained 90% of the empirical data variance. We showed that the relationship and dominance of the anomalous diffusion exponent resulted from a general mathematical relationship obtained from first-principles for any subdiffusion mechanism. Our derivation demonstrated that the generalized diffusion coefficient is not a measurable physical quantity and must be replaced by the length and time scales associated with the underlying mobility mechanisms. This led us to a fundamental reformulation of the classic subdiffusion equation, which calls for a reinterpretation of anomalous diffusion in physical systems. We also discussed how our results impact the characterization of microscopic particle diffusion in mucus and other hydrogels.

The actin cytoskeleton is an inherently disordered active system. the actomyosin cortex and reconstituted actomyosin systems are globally disordered, yet undergo transitions between distinct disordered states as parameters like motor and crosslinker concentration and filament length and rigidity change. In cells these changes are related to genetic mutations or differences in cell state and dictate fundamental biological processes. However, we dont have well established methods to detect and classify differences in disordered polymer networks. Image-based morphology techniques provide a non-invasive, high-throughput method of extracting information about a system. In this work we simulate biopolymer networks under varying conditions and develop and use morphological descriptors to construct trajectories in morphospace. Using statistical analysis we find that morphological descriptors are able to distinguish between different trajectories of the system, including differences not apparent to the eye. However, no single descriptor alone is able to capture all the differences in the simulated trajectories. Nematic order parameters typically perform the worst for our simulations while curvature and texture descriptors can collectively distinguish between dynamic trajectories. This work helps develop quantification of cytoskeleton dynamics for classification and data-driven modeling.

Microtubule (MT) polymerization is regulated by biochemical as well as physical factors such as macromolecular crowding. Crowding agents or crowdants affect MT elongation rates differently depending on crowdant size due to opposing ef-fects on polymerization: microviscosity reduces polymer elongation, while volume exclusion increases reaction rates by local concentration. In order to address how crowdant size and concentration collectively affect MT populations, we combine in vitro MT polymerization experiments with kinetic Monte Carlo simulations. Our experiments in bulk with nucleators validate decreasing MT elongation rates with increasing concentrations of small molecular weight crowdants in bulk assays and a corresponding increase for large crowdants. Kinetic Monte Carlo simulations can explain the result with packing fractions dependence of small as compared to large crowdants increasing microviscosity more dramatically. In contrast MT bulk polymerization rates in absence of nucleators increased with crowdant con-centration, irrespective of their size, with a corresponding decrease in the critical concentration. Microscopy of filament growth dynamics demonstrates that small crowdants result in shorter filaments in a concentration dependent manner, consis-tent with their role in reducing elongation rates, but this decrease is compensated by increased number of filaments. Large crowdants increase the filament numbers while elongation is slightly decreased. Our results provide evidence for MT nucle-ation being rate-limited and elongation diffusion limited, resulting in differences in the effect of crowdant sizes on nucleation and elongation. These results are of gen-eral relevance to understand physical effects of crowding on collective cytoskeletal polymerization dynamics.

Blood clots are essential biomaterials that prevent blood loss and provide a temporary scaffold for tissue repair. In their function, these materials must be capable of resisting mechanical forces from hemodynamic shear and contractile tension without rupture. Fibrin networks, the primary load-bearing element in blood clots, have unique nonlinear mechanical properties resulting from their hierarchical structure, which provides multiscale load bearing from fiber deformation to protein unfolding. Here, we study the fiber and molecular scale response of fibrin under shear and tensile loads in situ using a combination of fluorescence and vibrational (molecular) microscopy. Imaging protein fiber orientation and molecular vibrations, we find that fiber orientation and molecular changes in fibrin appear at much larger strains under shear compared to uniaxial tension. Orientation levels reached at 150% shear strain were reached already at 60% tensile strain, and molecular unfolding of fibrin was only seen at shear strains above 300%, whereas fibrin unfolding began already at 20% tensile strain. Moreover, shear deformation caused progressive changes in vibrational modes consistent with increased protofibril and fiber packing that were already present even at very low tensile deformation. Together with a bioinformatic analysis of the fibrinogen primary structure, we propose a scheme for the molecular response of fibrin from low to high deformation, which may relate to the teleological origin of its resistance to shear and tensile forces. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/205005v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@d72dfborg.highwire.dtl.DTLVardef@10bed75org.highwire.dtl.DTLVardef@12d33aorg.highwire.dtl.DTLVardef@1e9b40f_HPS_FORMAT_FIGEXP M_FIG C_FIG