Shaping membrane vesicles by adsorption of hinge-like nanoparticles.

The adsorption of particles onto fluid membranes can lead to membrane-mediated interactions between particles that promote their self-assembly and lead to changes in membrane morphology. However, in contrast with rigid particles, relatively little is known about deformable particles, which introduce additional complexities due to the mutual deformability of the particles and the membrane. Here, we use Monte Carlo simulations and umbrella sampling to investigate the equilibrium properties of hinge-like particles adsorbed on membrane vesicles by means of anisotropic, attractive interactions. We vary the hinge stiffness, adhesive area fraction, patterning of adhesive regions, and number of adsorbed particles. Depending on their properties, isolated particles can conform to the vesicle, induce invaginations of the membrane, or exhibit multistable behavior in which they sample distinct classes of configurations due to the interplay of particle and membrane deformations. With two adsorbed particles, the properties of the particles can be used to promote aggregation, bias the particles to different parts of the vesicle, or stabilize the coexistence of both cases. With multiple adsorbed particles, the number and type control their organization and collective impact on the vesicle, which can adopt shapes ranging from roughly spherical to dumbbell-like and multi-lobed. Our results highlight how modifying the mechanical properties and patterned adhesion of deformable particles, which is possible with DNA nanotechnology, influences their self-assembly and the resulting shapes of both the particles and vesicles.

Compartmental exchange regulates steady states and stochastic switching of a phosphorylation network.

The phosphoregulation of proteins with multiple phosphorylation sites is governed by biochemical reaction networks that can exhibit multistable behavior. However, the behavior of such networks is typically studied in a single reaction volume, while cells are spatially organized into compartments that can exchange proteins. In this work, we use stochastic simulations to study the impact of compartmentalization on a two-site phosphorylation network. We characterize steady states and fluctuation-driven transitions between them as a function of the rate of protein exchange between two compartments. Surprisingly, the average time spent in a state before stochastically switching to another depends nonmonotonically on the protein exchange rate, with the most frequent switching occurring at intermediate exchange rates. At sufficiently small exchange rates, the state of the system and mean switching time are controlled largely by fluctuations in the balance of enzymes in each compartment. This leads to negatively correlated states in the compartments. For large exchange rates, the two compartments behave as a single effective compartment. However, when the compartmental volumes are unequal, the behavior differs from a single compartment with the same total volume. These results demonstrate that exchange of proteins between distinct compartments can regulate the emergent behavior of a common signaling motif.

Particle Deformability Enables Control of Interactions between Membrane-Anchored Nanoparticles.

Nanoparticles adsorbed on a membrane can induce deformations of the membrane that give rise to effective interactions between the particles. Previous studies have focused primarily on rigid nanoparticles with fixed shapes. However, DNA origami technology has enabled the creation of deformable nanostructures with controllable shapes and mechanical properties, presenting new opportunities to modulate interactions between particles adsorbed on deformable surfaces. Here we use coarse-grained molecular dynamics simulations to investigate deformable, hinge-like nanostructures anchored to lipid membranes via cholesterol anchors. We characterize deformations of the particles and membrane as a function of the hinge stiffness. Flexible particles adopt open configurations to conform to a flat membrane, whereas stiffer particles induce deformations of the membrane. We further show that particles spontaneously aggregate and that cooperative effects lead to changes in their shape when they are close together. Using umbrella sampling methods, we quantify the effective interaction between two particles and show that stiffer hinge-like particles experience stronger and longer-ranged attraction. Our results demonstrate that interactions between deformable, membrane-anchored nanoparticles can be controlled by modifying mechanical properties of the particles, suggesting new ways to modulate the self-assembly of particles on deformable surfaces.

Propulsive cell entry diverts pathogens from immune degradation by remodeling the phagocytic synapse.

Phagocytosis is a critical immune function for infection control and tissue homeostasis. During phagocytosis, pathogens are internalized and degraded in phagolysosomes. For pathogens that evade immune degradation, the prevailing view is that virulence factors are required to disrupt the biogenesis of phagolysosomes. In contrast, we present here that physical forces from motile pathogens during cell entry divert them away from the canonical degradative pathway. This altered fate begins with the force-induced remodeling of the phagocytic synapse formation. We used the parasite Toxoplasma gondii as a model because live Toxoplasma actively invades host cells using gliding motility. To differentiate the effects of physical forces from virulence factors in phagocytosis, we employed magnetic forces to induce propulsive entry of inactivated Toxoplasma into macrophages. Experiments and computer simulations show that large propulsive forces hinder productive activation of receptors by preventing their spatial segregation from phosphatases at the phagocytic synapse. Consequently, the inactivated parasites are engulfed into vacuoles that fail to mature into degradative units, similar to the live motile parasite's intracellular pathway. Using yeast cells and opsonized beads, we confirmed that this mechanism is general, not specific to the parasite used. These results reveal new aspects of immune evasion by demonstrating how physical forces during active cell entry, independent of virulence factors, enable pathogens to circumvent phagolysosomal degradation.

Small RNA sequencing of field Culex mosquitoes identifies patterns of viral infection and the mosquito immune response.

Mosquito-borne disease remains a significant burden on global health. In the United States, the major threat posed by mosquitoes is transmission of arboviruses, including West Nile virus by mosquitoes of the Culex genus. Virus metagenomic analysis of mosquito small RNA using deep sequencing and advanced bioinformatic tools enables the rapid detection of viruses and other infecting organisms, both pathogenic and non-pathogenic to humans, without any precedent knowledge. In this study, we sequenced small RNA samples from over 60 pools of Culex mosquitoes from two major areas of Southern California from 2017 to 2019 to elucidate the virome and immune responses of Culex. Our results demonstrated that small RNAs not only allowed the detection of viruses but also revealed distinct patterns of viral infection based on location, Culex species, and time. We also identified miRNAs that are most likely involved in Culex immune responses to viruses and Wolbachia bacteria, and show the utility of using small RNA to detect antiviral immune pathways including piRNAs against some pathogens. Collectively, these findings show that deep sequencing of small RNA can be used for virus discovery and surveillance. One could also conceive that such work could be accomplished in various locations across the world and over time to better understand patterns of mosquito infection and immune response to many vector-borne diseases in field samples.

Topographical analysis of immune cell interactions reveals a biomechanical signature for immune cytolysis.

Immune cells live intensely physical lifestyles characterized by structural plasticity, mechanosensitivity, and force exertion. Whether specific immune functions require stereotyped patterns of mechanical output, however, is largely unknown. To address this question, we used super-resolution traction force microscopy to compare cytotoxic T cell immune synapses with contacts formed by other T cell subsets and macrophages. T cell synapses were globally and locally protrusive, which was fundamentally different from the coupled pinching and pulling of macrophage phagocytosis. By spectrally decomposing the force exertion patterns of each cell type, we associated cytotoxicity with compressive strength, local protrusiveness, and the induction of complex, asymmetric interfacial topographies. These features were further validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, direct imaging of synaptic secretory events, and in silico analysis of interfacial distortion. We conclude that T cell-mediated killing and, by implication, other effector responses are supported by specialized patterns of efferent force.

Propulsive cell entry diverts pathogens from immune degradation by remodeling the phagocytic synapse.

Phagocytosis is a critical immune function for infection control and tissue homeostasis. This process is typically described as non-moving pathogens being internalized and degraded in phagolysosomes. For pathogens that evade immune degradation, the prevailing view is that virulence factors that biochemically disrupt the biogenesis of phagoslysosomes are required. In contrast, here we report that physical forces exerted by pathogens during cell entry divert them away from the canonical phagolysosomal degradation pathway, and this altered intracellular fate is determined at the time of phagocytic synapse formation. We used the eukaryotic parasite Toxoplasma gondii as a model because live Toxoplasma uses gliding motility to actively invade into host cells. To differentiate the effect of physical forces from that of virulence factors in phagocytosis, we developed a strategy that used magnetic forces to induce propulsive entry of inactivated Toxoplasma into macrophage cells. Experiments and computer simulations collectively reveal that large propulsive forces suppress productive activation of receptors by hindering their spatial segregation from phosphatases at the phagocytic synapse. Consequently, the inactivated parasites, instead of being degraded in phagolysosomes, are engulfed into vacuoles that fail to mature into degradative units, following an intracellular pathway strikingly similar to that of the live motile parasite. Using opsonized beads, we further confirmed that this mechanism is general, not specific to the parasite used. These results reveal previously unknown aspects of immune evasion by demonstrating how physical forces exerted during active cell entry, independent of virulence factors, can help pathogens circumvent phagolysosomal degradation.

Organization and dynamics of cross-linked actin filaments in confined environments.

The organization of the actin cytoskeleton is impacted by the interplay between physical confinement, features of cross-linking proteins, and deformations of semiflexible actin filaments. Some cross-linking proteins preferentially bind filaments in parallel, although others bind more indiscriminately. However, a quantitative understanding of how the mode of binding influences the assembly of actin networks in confined environments is lacking. Here we employ coarse-grained computer simulations to study the dynamics and organization of semiflexible actin filaments in confined regions upon the addition of cross-linkers. We characterize how the emergent behavior is influenced by the system shape, the number and type of cross-linking proteins, and the length of filaments. Structures include isolated clusters of filaments, highly connected filament bundles, and networks of interconnected bundles and loops. Elongation of one dimension of the system promotes the formation of long bundles that align with the elongated axis. Dynamics are governed by rapid cross-linking into aggregates, followed by a slower change in their shape and connectivity. Cross-linking decreases the average bending energy of short or sparsely connected filaments by suppressing shape fluctuations. However, it increases the average bending energy in highly connected networks because filament bundles become deformed, and small numbers of filaments exhibit long-lived, highly unfavorable configurations. Indiscriminate cross-linking promotes the formation of high-energy configurations due to the increased likelihood of unfavorable, difficult-to-relax configurations at early times. Taken together, this work demonstrates physical mechanisms by which cross-linker binding and physical confinement impact the emergent behavior of actin networks, which is relevant both in cells and in synthetic environments.

Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles.

Cell-free protein synthesis is an important tool for studying gene expression and harnessing it for applications. In cells, gene expression is regulated in part by the spatial organization of transcription and translation. Unfortunately, current cell-free approaches are unable to control the organization of molecular components needed for gene expression, which limits the ability to probe and utilize its effects. Here, we show, using complementary computational and experimental approaches, that macromolecular crowding can be used to control the spatial organization and translational efficiency of gene expression in cell-sized vesicles. Computer simulations and imaging experiments reveal that, as crowding is increased, DNA plasmids become localized at the inner surface of vesicles. Ribosomes, in contrast, remain uniformly distributed, demonstrating that crowding can be used to differentially organize components of gene expression. We further carried out cell-free protein synthesis reactions in cell-sized vesicles and quantified mRNA and protein abundance. At sufficiently high levels of crowding, we observed localization of mRNA near vesicle surfaces, a decrease in translational efficiency and protein abundance, and anomalous scaling of protein abundance as a function of vesicle size. These results are consistent with high levels of crowding causing altered spatial organization and slower diffusion. Our work demonstrates a straightforward way to control the organization of gene expression in cell-sized vesicles and provides insight into the spatial regulation of gene expression in cells.

Membrane-mediated interactions between hinge-like particles.

Adsorption of nanoparticles on a membrane can give rise to interactions between particles, mediated by membrane deformations, that play an important role in self-assembly and membrane remodeling. Previous theoretical and experimental research has focused on nanoparticles with fixed shapes, such as spherical, rod-like, and curved nanoparticles. Recently, hinge-like DNA origami nanostructures have been designed with tunable mechanical properties. Inspired by this, we investigate the equilibrium properties of hinge-like particles adsorbed on an elastic membrane using Monte Carlo and umbrella sampling simulations. The configurations of an isolated particle are influenced by competition between bending energies of the membrane and the particle, which can be controlled by changing adsorption strength and hinge stiffness. When two adsorbed particles interact, they effectively repel one another when the strength of adhesion to the membrane is weak. However, a strong adhesive interaction induces an effective attraction between the particles, which drives their aggregation. The configurations of the aggregate can be tuned by adjusting the hinge stiffness: tip-to-tip aggregation occurs for flexible hinges, whereas tip-to-middle aggregation also occurs for stiffer hinges. Our results highlight the potential for using the mechanical features of deformable nanoparticles to influence their self-assembly when the particles and membrane mutually influence one another.

Adsorption of semiflexible polymers in crowded environments.

Macromolecular crowding is a feature of cellular and cell-free systems that, through depletion effects, can impact the interactions of semiflexible biopolymers with surfaces. In this work, we use computer simulations to study crowding-induced adsorption of semiflexible polymers on otherwise repulsive surfaces. Crowding particles are modeled explicitly, and we investigate the interplay between the bending stiffness of the polymer and the volume fraction and size of crowding particles. Adsorption to flat surfaces is promoted by stiffer polymers, smaller crowding particles, and larger volume fractions of crowders. We characterize transitions from non-adsorbed to partially and strongly adsorbed states as a function of bending stiffness. The crowding-induced transitions occur at smaller values of the bending stiffness as the volume fraction of crowders increases. Concomitant effects on the size and shape of the polymer are reflected by crowding- and stiffness-dependent changes to the radius of gyration. For various polymer lengths, we identify a critical crowding fraction for adsorption and analyze its scaling behavior in terms of polymer stiffness. We also consider crowding-induced adsorption in spherical confinement and identify a regime in which increasing the bending stiffness induces desorption. The results of our simulations shed light on the interplay of crowding and bending stiffness on the spatial organization of biopolymers in encapsulated cellular and cell-free systems.

Crowding-induced interactions of ring polymers.

Macromolecular crowding and the presence of surfaces can significantly impact the spatial organization of biopolymers. While the importance of crowding-induced depletion interactions in biology has been recognized, much remains to be understood about the effect of crowding on biopolymers such as DNA plasmids. A fundamental problem highlighted by recent experiments is to characterize the impact of crowding on polymer-polymer and polymer-surface interactions. Motivated by the need for quantitative insight, we studied flexible ring polymers in crowded environments using Langevin dynamics simulations. The simulations demonstrated that crowding can lead to compaction of isolated ring polymers and enhanced interactions between two otherwise repulsive polymers. Using umbrella sampling, we determined the potential of mean force (PMF) between two ring polymers as a function of their separation distance at different volume fractions of crowding particles, φ. An effective attraction emerged at φ≈ 0.4, which is similar to the degree of crowding in cells. Analogous simulations showed that crowding can lead to strong adsorption of a ring polymer to a wall, with an effective attraction to the wall emerging at a smaller volume fraction of crowders (φ≈ 0.2). Our results reveal the magnitude of depletion interactions in a biologically-inspired model and highlight how crowding can be used to tune interactions in both cellular and cell-free systems.

Protein Molecular Dynamics Simulations with Approximate QM: What Can We Learn?

Classical force fields are essential for computer simulations of proteins and are typically parameterized to reproduce secondary and tertiary structure of isolated proteins. However, while protein-protein interactions are ubiquitous in nature, they are not considered in parameterization efforts and are far less understood than isolated proteins. A better characterization of intermolecular interactions is widely recognized as a key to revolutionizing drug and therapeutic developments with high-throughput computational screening. Urgently needed is a critical assessment of the performance of modern protein force fields against first-principles electronic structure methods and experiments. In a daring step toward this goal, we here describe a comparison of peptide folding dynamics as predicted by a molecular mechanics force field on the one hand and by an approximate electronic structure quantum mechanical (QM) method based on density-functional tight-binding (DFTB) on the other. We further compare the dynamics from straightforward DFTB simulations with a near-linear scaling version of DFTB for massively parallel computation based on the fragment molecular orbital (FMO-DFTB) method. We illustrate differences between the phenomenology of the folding dynamics from these three methods for a small model peptide, as well as charge polarization and dynamic fluctuations, point out possible correlations and implications for force field developers, and discuss the lessons learned that might become applicable to future predictive high-throughput computer screening for personalized neoantigen cancer therapy.

Mechanical feedback enables catch bonds to selectively stabilize scanning microvilli at T-cell surfaces.

T-cells use microvilli to search the surfaces of antigen-presenting cells for antigenic ligands. The active motion of scanning microvilli provides a force-generating mechanism that is intriguing in light of single-molecule experiments showing that applied forces increase the lifetimes of stimulatory receptor-ligand bonds (catch-bond behavior). In this work, we introduce a theoretical framework to explore the motion of a microvillar tip above an antigen-presenting surface when receptors on the tip stochastically bind to ligands on the surface and dissociate from them in a force-dependent manner. Forces on receptor-ligand bonds impact the motion of the microvillus, leading to feedback between binding and microvillar motion. We use computer simulations to show that the average microvillar velocity varies in a ligand-dependent manner; that catch bonds generate responses in which some microvilli almost completely stop, while others move with a broad distribution of velocities; and that the frequency of stopping depends on the concentration of stimulatory ligands. Typically, a small number of catch bonds initially immobilize the microvillus, after which additional bonds accumulate and increase the cumulative receptor-engagement time. Our results demonstrate that catch bonds can selectively slow and stabilize scanning microvilli, suggesting a physical mechanism that may contribute to antigen discrimination by T-cells.

First passage of molecular motors on networks of cytoskeletal filaments.

Molecular motors facilitate intracellular transport through a combination of passive motion in the cytoplasm and active transport along cytoskeletal filaments. Although the motion of motors on individual filaments is often well characterized, it remains a challenge to understand their transport on networks of filaments. Here we use computer simulations of a stochastic jump process to determine first-passage times (FPTs) of a molecular motor traversing an interval containing randomly distributed filaments of fixed length. We characterize the mean first-passage time (MFPT) as a function of the number and length of filaments. Intervals containing moderate numbers of long filaments lead to the largest MFPTs with the largest relative standard deviation; in this regime, some filament configurations lead to anomalously large FPTs due to spatial regions where motors become trapped for long times. For specific filament configurations, we systematically reverse the directionality of single filaments and determine the MFPT of the perturbed configuration. Surprisingly, altering a single filament can dramatically impact the MFPT, and filaments leading to the largest changes are commonly found in different regions than the traps. We conclude by analyzing the mean square displacement of motors in unconfined systems with a large density of filaments and show that they behave diffusively at times substantially less than the MFPT to traverse the interval. However, the effective diffusion coefficient underestimates the MFPT across the bounded interval, emphasizing the importance of local configurations of filaments on first-passage properties.

A Cdc42 GEF, Gef1, through endocytosis organizes F-BAR Cdc15 along the actomyosin ring and promotes concentric furrowing.

During cytokinesis, fission yeast coordinates actomyosin ring constriction with septum ingression, resulting in concentric furrow formation by a poorly defined mechanism. We report that Schizosaccharomyces pombe cells lacking the Cdc42 activator Gef1, combined with an activated allele of the formin, Cdc12, display non-concentric furrowing. Non-concentrically furrowing cells display uneven distribution of the scaffold Cdc15 along the ring. This suggests that, after ring assembly, uniform Cdc15 distribution along the ring enables proper furrow formation. We find that, after assembly, Cdc15 is recruited to the ring in an Arp2/3 complex-dependent manner and is decreased in the activated cdc12 mutant. Cdc15 at cortical endocytic patches shows increased levels and extended lifetimes in gef1 and activated cdc12 mutants. We hypothesize endocytosis helps recruit Cdc15 to assembled rings; uneven Cdc15 distribution at the ring occurs when endocytic patches contain increased Cdc15 levels and the patch-association rate is slow. Based on this, we developed a mathematical model that captures experimentally observed Cdc15 distributions along the ring. We propose that, at the ring, Gef1 and endocytic events promote uniform Cdc15 organization to enable proper septum ingression and concentric furrow formation.

A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration.

Misregulation of the signaling axis formed by the receptor tyrosine kinase (RTK) EphA2 and its ligand, ephrinA1, causes aberrant cell-cell contacts that contribute to metastasis. Solid tumors are characterized by an acidic extracellular medium. We intend to take advantage of this tumor feature to design new molecules that specifically target tumors. We created a novel pH-dependent transmembrane peptide, TYPE7, by altering the sequence of the transmembrane domain of EphA2. TYPE7 is highly soluble and interacts with the surface of lipid membranes at neutral pH, while acidity triggers transmembrane insertion. TYPE7 binds to endogenous EphA2 and reduces Akt phosphorylation and cell migration as effectively as ephrinA1. Interestingly, we found large differences in juxtamembrane tyrosine phosphorylation and the extent of EphA2 clustering when comparing TYPE7 with activation by ephrinA1. This work shows that it is possible to design new pH-triggered membrane peptides to activate RTK and gain insights on its activation mechanism.

Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns.

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting-the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increase in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA ("spatial noise") that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. These results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.

Shaping membrane vesicles by adsorption of a semiflexible polymer.

The adsorption of polymers onto fluid membranes is a problem of fundamental interest in biology and soft materials, in part because the flexibility of membranes can lead to nontrivial coupling between polymer and membrane configurations. Here, we use Monte Carlo computer simulations to study the adsorption of a semiflexible polymer onto a fluid membrane vesicle. Polymer adsorption can significantly impact both the vesicle and polymer shapes, and we identify distinct classes of configurations that emerge as a function of polymer persistence length, membrane bending rigidity, adsorption strength, and vesicle size. Large-scale deformations of the vesicle include invaginations of the membrane that internalize the polymer in a membrane bud. The buds range from disk-like shapes surrounding a collapsed polymer to tubular deformations enveloping rod-like polymers. For small vesicles, polymer adsorption also induces dumbbell-like vesicle shapes with a narrow membrane constriction circled by the polymer. Vesicles with sufficiently small or large bending rigidities adopt configurations similar to those without the polymer present. We further characterize statistical properties of the membrane and polymer configurations and identify distinct classes of polymer configurations that emerge within membrane buds. Analysis of idealized polymer-membrane configurations provides additional insight into transitions between bud shapes.

Catch Bonds at T Cell Interfaces: Impact of Surface Reorganization and Membrane Fluctuations.

Catch bonds are characterized by average lifetimes that initially increase with increasing tensile force. Recently, they have been implicated in T cell activation, where small numbers of antigenic receptor-ligand bonds at a cell-cell interface can stimulate a T cell. Here, we use computational methods to investigate small numbers of bonds at the interface between two membranes. We characterize the time-dependent forces on the bonds in response to changes in the membrane shape and the organization of other surface molecules. We then determine the distributions of bond lifetimes using recent force-dependent lifetime data for T cell receptors bound to various ligands. Strong agonists, which exhibit catch bond behavior, are markedly more likely to remain intact than an antagonist whose average lifetime decreases with increasing force. Thermal fluctuations of the membrane shape enhance the decay of the average force on a bond, but also lead to fluctuations of the force. These fluctuations promote bond rupture, but the effect is buffered by catch bonds. When more than one bond is present, the bonds experience reduced average forces that depend on their relative positions, leading to changes in bond lifetimes. Our results highlight the importance of force-dependent binding kinetics when bonds experience time-dependent and fluctuating forces, as well as potential consequences of collective bond behavior relevant to T cell activation.