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.

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.

Single-cell topographical profiling of the immune synapse reveals a biomechanical signature of cytotoxicity.

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.

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.

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.