The neutrophil is a key player in immunity, and its activities are essential for the resolution of infections. Neutrophil-pathogen interactions usually trigger a large arsenal of antimicrobial measures that leads to the highly efficient killing of pathogens. In neutrophils, the phagocytic process, including the formation and maturation of the phagosome, is in many respects very different from that in other phagocytes. Although the complex mechanisms that coordinate the membrane traffic, oxidative burst, and release of granule contents required for the microbicidal activities of neutrophils are not completely understood, it is evident that they are unique and differ from those in macrophages. Neutrophils exhibit more rapid rates of phagocytosis and higher intensity of oxidative respiratory response than do macrophages. The phagosome maturation pathway in macrophages, which is linked to the endocytic pathway, is replaced in neutrophils by the rapid delivery of preformed granules to nonacidic phagosomes. This review describes the plasticity and dynamics of the phagocytic process with a special focus on neutrophil phagosome maturation.

For a cell to move forward it must convert chemical energy into mechanical propulsion. Force produced by actin polymerization can generate traction across the plasma membrane by transmission through integrins to their ligands. However, the role this force plays in integrin activation is unknown. Here we show that integrin activity and cytoskeletal dynamics are reciprocally linked, where actin-dependent force itself appears to regulate integrin activity. We generated fluorescent tension-sensing constructs of integrin αLβ2 (LFA-1) to visualize intramolecular tension during cell migration. Using quantitative imaging of migrating T cells, we correlate tension in the αL or β2 subunit with cell and actin dynamics. We find that actin engagement produces tension within the β2 subunit to induce and stabilize an active integrin conformational state and that this requires intact talin and kindlin motifs. This supports a general mechanism where localized actin polymerization can coordinate activation of the complex machinery required for cell migration.

Small interfering RNAs (siRNAs) are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes. However, endosomal entrapment of siRNA is a key hurdle for most delivery strategies, limiting the therapeutic effect. Here, we use live-cell microscopy and cytosolic galectin-9 as a sensor of membrane damage, to probe fundamental properties of endosomal escape of cholesterol-conjugated siRNA induced by endosome-disrupting compounds. We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to ∼47-fold in tumor cells. Still, mismatch between siRNA-containing and drug-targeted endolysosomal compartments limits siRNA activity improvement. We also show widespread endosomal damage in macroscopic tumor spheroids after small molecule treatment, substantially improving siRNA delivery and knockdown throughout the spheroid. We believe the strategy to characterize endosomal escape presented here will be widely applicable, facilitating efforts to improve delivery of siRNA and other nucleic acid-based therapeutics.

Significance: Integrins are adhesion receptors linking cells to their environment, which function as sensors of physical and chemical information to regulate development, immune response, and vascular function. How integrins receive and transduce directional forces including flow or tissue tension has remained elusive. We used polarization-based microscopy techniques to discover that activated αVβ3 integrins are aligned with one another in focal adhesions in migrating fibroblasts. Integrin coalignment is sensitive to mechanical resistance of its ligand and coupling to a dynamic F-actin cytoskeleton, consistent with the “cytoskeleton force model” for integrin activation. Our work suggests that activated integrins are actively ordered at the molecular scale by cellular forces, which may underlie their ability to sense directional forces in their environment to mediate critical functions.

Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.

Lipid nanoparticles (LNPs) are clinically approved for mRNA-based vaccines and liver-targeted siRNA delivery. However, poor nucleic acid delivery efficiency limits their application in extrahepatic tissues and tumors. Here, using live-cell and super-resolution microscopy, we identify multiple distinct steps of inefficiencies in the cytosolic delivery of both siRNA and mRNA cargoes. Membrane damages marked by galectin recruitment are conducive to cytosolic RNA release, whereas membrane perturbations recruiting the ESCRT machinery do not permit endosomal escape. Notably, only a small fraction of RNA is released from galectin-marked endosomes and, unexpectedly, many damaged endosomes contain no detectable RNA cargo. Using LNPs with both fluorescently labeled ionizable lipid and RNA, we show that these components segregate during endosomal sorting – both within single endosomes and across endosomal compartments. Finally, we visualize localized ionizable lipid enrichment in endosomal membranes and membrane damage in direct proximity to siRNA-LNPs tethered to luminal vesicle membranes. Taken together, our findings reveal multiple mechanistic barriers limiting intracellular RNA delivery by LNPs.