Article Title: Phagocytic “teeth” and myosin-II “jaw” power target constriction during phagocytosis
Figure Lengend Snippet: Arp2/3-mediated actin polymerization and myosin-II have distinct roles in phagocytic force generation and progression. a, Confocal images of drug-treated fixed RAW cells phagocytosing DAAM-particles functionalized with AF488-Cadaverine, BSA and anti-BSA IgG. Cells were treated with DMSO, CK666 (150 μM), Blebbistatin (15 μM) and SMIFH2 (10 μM) for 30 minutes prior to phagocytic challenge. Each target is approximately 60% engulfed. Fixed cells were stained for F-actin, and particles were labelled with a fluorescent secondary antibody to reveal the exposed surface. Left column: composite maximum intensity projections (MIP) of confocal z-stacks, 2 nd -3 rd column: single confocal slices through particle centroid. Scale bar, 5μm. b, Particle shape reconstructions from a, revealing detailed target deformations and localization of F-actin over the particle surface. Stars mark the base of the phagocytic cup, and the phagocytic axis is horizontal. Scale bars, 3 μm. c, Normal and shear stresses exerted on the target. Negative normal forces denote (inward) compressive forces. d, Average deformation and F-actin intensity profiles along the phagocytic axis to the cup rim. Signals were first processed on a per-particle basis by averaging over the surface along the phagocytic targets in 30 bins. Targets before 40% engulfment were excluded. e, f, Violin plots of all measured particles, showing individual phagocytic events (colored markers), mean (black cross) and median (dashed line). e, F-actin peak intensity and width. f, F-actin intensity in the cup (behind the rim), measured right (3μm) behind the main peak for each particle. g, Upper panel, cumulative distribution function of the engulfment stage of randomly selected phagocytic events (n = 68, 63, 73 55 respectively) from 3 independent experiments. Two sample Kolmogorov-Smirnov test was used (p = 0.016*). Lower panel, fraction late-stage cups. Error bars indicate st.d. estimated by treating phagocytosis as a Bernoulli process. Fisher’s exact test was used to compare fractions (p = 1.9×10 −4 )***. h, Sphericity and i, constriction magnitude of DAAM particle changes with phagocytic progression upon drug treatment. Colored markers indicate individual events, black lines indicate averages within 5 bins. Right column, violin plots of all events. Marker and line styles as in e. All statistical tests were two-side Wilcoxon rank sum test comparing with the DMSO control (gray) over the same bin with significance levels: p
Article Snippet: Phagocytosis assay DAAM particles were washed 3X in sterile PBS and opsonized with rabbit anti-BSA antibody (MP Biomedicals, 0865111) for 1 h at room temperature.
Techniques: Staining, Marker