FIGURE 2:
Phagocytosis by BMDMs is gentle and involves local pushing at the cup base. (A) Confocal images of fixed BMDMs phagocytosing soft (∼13 μm, 0.3 kPa) deformable acrylamide–coacrylic acid microparticles (DAAMPs) functionalized with IgG, and AF488-cadaverine for visualization. Cells were stained for F-actin and labeled with a fluorescent secondary antibody to reveal the exposed surface of the target. Left two columns: composite maximum intensity projections (MIPs) of confocal stacks; third to fifth column: single confocal slices (xy). (B) 3D shape reconstructions of DAAMPs in A revealing detailed target deformations induced during phagocytosis and localization of F-actin over the particle surface. (C) Target sphericity indicating total target deviation from a spherical shape, where 1 would be an undeformed perfect sphere. (D) Schematic representation of phagocytic parameterization. Normalized cup position indicates the relative position along the phagocytic axis between the cup base (0) and 1. (E) Direction of the phagocytic axis, where 0° indicates straight up from and 90° parallel to the coverslip. (F) Average profiles of target deformation and F-actin intensity along the phagocytic axis, where 0 and 1 are the cup base and rim, respectively. Signals were first averaged in rings over the surface along the phagocytic axis on a per-particle basis. Only targets beyond 40% engulfment were included (19 out of 30 events). (G) Relative elongation of particles undergoing phagocytosis. Wilcoxon signed rank test for differences with 1 (no compression or elongation) were performed for each sample, showing target compression for 0.3 (p = 0.047) and 1.4 kPa particles (p = 0.002) for BMDMs, and target elongation (p = 2 × 10−7) for RAW macrophages. (H) Number and localization of actin-containing protrusive spots per cup. All violin plots show individual phagocytic events (colored circles), mean (black cross), and median (dashed line), and all error bars indicate standard error of the mean. Unless otherwise indicated, statistical tests were two-sided Wilcoxon rank sum test with *, p < 0.05 and ***, p < 0.001. Data on RAW macrophages was taken from previously published work (Vorselen et al., 2021).