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The organization of cytoskeletal elements is pivotal for coordinating intracellular transport in eukaryotic cells. Several quantitative measures based on image analysis have been proposed to characterize morphometric features of fluorescently labeled actin networks. While helpful in detecting differences in actin organization between treatments or genotypes, the accuracy of these measures could not be rigorously assessed due to a lack of ground-truth data to which they could be compared. To overcome this limitation, we utilized coarse-grained computer simulations of actin filaments and cross-linkers to generate synthetic actin networks with varying levels of bundling. We converted the simulated networks into pseudofluorescence images similar to images obtained using confocal microscopy. Using both published and novel analysis procedures, we extracted a series of morphometric parameters and benchmarked them against analogous measures based on the ground-truth actin configurations. Our analysis revealed a set of parameters that reliably reports on actin network density, orientation, ordering, and bundling. Application of these morphometric parameters to root epidermal cells of Arabidopsis thaliana revealed subtle changes in network organization between wild-type and mutant cells. This work provides robust measures that can be used to quantify features of actin networks and characterize changes in actin organization for different experimental conditions. 

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. 

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. 

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. 

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 parasiteToxoplasma gondiias a model because liveToxoplasmaactively 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 inactivatedToxoplasmainto 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. 

Immune cells have intensely physical lifestyles characterized by structural plasticity and force exertion. To investigate whether specific immune functions require stereotyped mechanical outputs, we used super-resolution traction force microscopy to compare the immune synapses formed by cytotoxic T cells with contacts formed by other T cell subsets and by macrophages. T cell synapses were globally compressive, which was fundamentally different from the pulling and pinching associated with macrophage phagocytosis. Spectral decomposition of force exertion patterns from each cell type linked cytotoxicity to compressive strength, local protrusiveness, and the induction of complex, asymmetric topography. These features were validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, live imaging of synaptic secretion, and in silico analysis of interfacial distortion. Synapse architecture and force exertion were sensitive to target stiffness and size, suggesting that the mechanical potentiation of killing is biophysically adaptive. We conclude that cellular cytotoxicity and, by implication, other effector responses are supported by specialized patterns of efferent force. 

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.