Steven M. Abel

Understanding how nonenveloped viruses breach host cell membranes is critical for developing strategies to block viral entry, a key step in infection. Despite extensive study, how viral capsids and host lipid membranes dynamically cooperate during membrane penetration remains poorly defined. Here, using reovirus as a model nonenveloped virus and planar model membranes, we identify previously unrecognized non-Brownian membrane motions of infectious subvirion particles (ISVPs) by single-virus tracking. We then integrate experiments with computational modeling to dissect the stepwise, processive capsid–membrane interactions encoded in these distinct dynamics. We show that ISVP motion transitions from an initial phase of directed translocation to progressively confined diffusion. This behavior is consistent with a multistep entry mechanism in which initial capsid–membrane contact triggers release of the membrane-active μ1N peptide. Together, our data and simulations support a model in which membrane-inserted μ1N may accumulate locally to create retention sites that promote further virus adsorption and progressively restrict particle mobility. By directly visualizing these motion signatures, we resolve transient and cooperative capsid–membrane interactions that are difficult to capture using conventional biochemical approaches. Together, these findings provide new insight into early membrane penetration events of nonenveloped viruses.

Spatial variations in stiffness regulate polymer collapse and adsorption driven by depletion interactions in crowded environments.

Understanding how non-enveloped viruses breach host cell membranes is critical for developing strategies to block viral entry, a key step in infection. Despite extensive study, how viral capsids and host lipid membranes dynamically cooperate during membrane penetration remains poorly defined. Here, using reovirus as a model non-enveloped virus and planar model membranes, we identify previously unrecognized non-Brownian membrane motions of infectious subvirion particles (ISVPs) by single-virus tracking. We then integrate experiments with computational modeling to dissect the stepwise, processive capsid–membrane interactions encoded in these distinct dynamics. We show that ISVP motion transitions from an initial phase of directed translocation to progressively confined diffusion. This behavior reflects a multistep entry mechanism in which initial capsid–membrane contact triggers release of the membrane-active μ1N peptide. As μ1N accumulates within the bilayer, it generates membrane-associated viral retention sites that promote further virus adsorption and increasingly constrain particle mobility. By directly visualizing these motion signatures, we resolve transient and cooperative capsid–membrane interactions that are difficult to capture using conventional biochemical approaches. Together, these findings provide new insight into early membrane penetration events of non-enveloped viruses. SIGNIFICANCE STATEMENT: Non-enveloped viruses must penetrate host cell membranes to initiate infection, yet the dynamic mechanisms by which viral capsids engage and remodel lipid membranes remain poorly understood. By directly visualizing the motion of individual reovirus particles on model membranes, we uncover previously unrecognized non-Brownian dynamics that encode stepwise capsid–membrane interactions during viral entry. Integrating single-virus tracking with computational modeling reveals how peptide-mediated membrane remodeling feeds back to regulate viral engagement. This work provides the first direct visualization of processive, stepwise capsid–membrane interactions during non-enveloped virus entry and establishes viral particle dynamics as a quantitative readout for dissecting membrane penetration mechanisms.

Macromolecular crowding can significantly impact the behavior of biopolymers, with crowding-induced depletion interactions influencing both the conformations and surface adsorption of individual polymers. Although previous studies have explored the influence of homogeneous polymer stiffness in crowded conditions, biomolecules such as DNA can exhibit sequence-dependent stiffness, and DNA origami nanoparticles can be designed with alternating stiff and flexible domains. In this work, we use Langevin dynamics simulations to characterize how nonuniform bending stiffness modulates the conformations and adsorption of polymers in crowded environments. By systematically varying the relative length and arrangement of flexible and semiflexible domains along a linear chain, we show that increasing osmotic pressure leads to a pattern-dependent collapse of the polymer, as revealed by a decrease in the radius of gyration. In general, large flexible regions promote polymer collapse, although flexible domains separating extended semi-flexible regions can facilitate their contact, leading to stable folded conformations. When a surface is present, large semiflexible domains promote adsorption, and the pattern of stiffness can be used to control the adsorption threshold. Our findings provide insight into the impact of spatially varying stiffness on the behavior of polymers in crowded environments, highlighting mechanisms relevant to biopolymers and deformable nanoparticles in both cellular and cell-free contexts.

Lipid droplets (LDs) are ubiquitous neutral lipid storage organelles that form at discrete subdomains in the ER bilayer. The assembly of these ER subdomains and the mechanism by which proteins are recruited to them is poorly understood. Here, we investigate the spatiotemporal distribution of Pex30 at the ER-LD membrane contact sites (MCSs). Pex30, an ER membrane–shaping protein, has a reticulon homology domain, a dysferlin (DysF) domain, and a Duf4196 domain. Deletion of SEI1, which codes for seipin, a highly conserved protein required for LD biogenesis, results in accumulation of Pex30 and phosphatidic acid (PA) at ER-LD contact sites. We show that PA recruits Pex30 at ER subdomains by binding to the DysF domain. The distribution of Pex30 as well as PA is also affected by phosphatidylcholine (PC) levels. We propose that PA regulates the spatiotemporal distribution of Pex30 at ER subdomains that plays a critical role in driving the formation of LDs in the ER membrane.

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.

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.

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.

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 crosslinkers to generate synthetic actin networks with varying levels of bundling. We converted the simulated networks into pseudo-fluorescence 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.