The spatial resolution limit of phagocytosis.

Antibody-opsonized bacteria interact with Fc receptors in macrophages and trigger signaling cascades, which induce phagocytosis. The signaling pathways ultimately lead to actin polymerization that induces the protrusion of the membrane around the bacterium until it is completely engulfed. Although many proteins involved in the phagocytic cup formation have already been identified, it is still unclear how far the initial stimulus created by the bacterium-cell contact propagates in the cell. We hypothesize that this spreading distance is closely related to the spatial resolution limit of phagocytosis, the smallest distance in which two stimuli can be differentiated. Here, we probe this resolution limit by using holographic optical tweezers to attach pairs of immunoglobulin G-coated polystyrene microparticles (as models for opsonized bacteria) to murine macrophages in distances ranging from zero to several micrometers. By using 2-µm-sized particles, we found that the particles can be internalized jointly into one phagosome if they are attached to the cell very close together, but that they are taken up separately if they are attached far from each other. To explain this, we developed a model of the signaling process, which predicts the probabilities for separate uptake for different particle sizes and distances using cellular parameters including the average receptor distance. We tested the model by measuring the separate uptake probabilities for particles with a diameter of 1 to 3 µm and for cells with reduced numbers of Fcγ receptors and found very good agreement. Our model shows that the phagocytic uptake behavior can be explained by assuming an effective phagocytic signaling range of about 500 nm. Interestingly, this value corresponds to the lower size limit of phagocytosis. Our work provides quantitative access to spatial parameters of cellular signaling during phagocytosis and thereby contributes to a more quantitative understanding of cellular information processing.

Using blinking optical tweezers to study cell rheology during initial cell-particle contact.

Phagocytosis is an important part of innate immunity and describes the engulfment of bacteria and other extracellular objects on the micrometer scale. The protrusion of the cell membrane around the bacteria during this process is driven by a reorganization of the actin cortex. The process has been studied on the molecular level to great extent during the past decades. However, a deep, fundamental understanding of the mechanics of the process is still lacking, in particular because of a lack of techniques that give access to binding dynamics below the optical resolution limit and cellular viscoelasticity at the same time. In this work, we propose a technique to characterize the mechanical properties of cells in a highly localized manner and apply it to investigate the early stages of phagocytosis. The technique can simultaneously resolve the contact region between a cell and an external object (in our application, a phagocytic target) even below the optical resolution limit. We used immunoglobulin-G-coated microparticles with a size of 2 µm as a model system and attached the particles to the macrophages with holographic optical tweezers. By switching the trap on and off, we were able to measure the rheological properties of the cells in a time-resolved manner during the first few minutes after attachment. The measured viscoelastic cellular response is consistent with power law rheology. The contact radius between particle and cell increased on a timescale of ∼30 s and converged after a few minutes. Although the binding dynamics are not affected by cytochalasin D, we observed an increase of the cellular compliance and a significant fluidization of the cortex after addition of cytochalasin D treatment. Furthermore, we report upper boundaries for the length- and timescale, at which cortical actin has been hypothesized to depolymerize during early phagocytosis.

Wrapping of Microparticles by Floppy Lipid Vesicles.

Lipid membranes, the barrier defining living cells and many of their subcompartments, bind to a wide variety of nano- and micrometer sized objects. In the presence of strong adhesive forces, membranes can strongly deform and wrap the particles, an essential step in crossing the membrane for a variety of healthy and disease-related processes. A large body of theoretical and numerical work has focused on identifying the physical properties that underly wrapping. Using a model system of micron-sized colloidal particles and giant unilamellar lipid vesicles with tunable adhesive forces, we measure a wrapping phase diagram and make quantitative comparisons to theoretical models. Our data are consistent with a model of membrane-particle interactions accounting for the adhesive energy per unit area, membrane bending rigidity, particle size, and vesicle radius.