Double knockout of Myo1e and Myo1f in macrophages markedly reduced uptake of IgG-coated beads and caused premature formation of the phagocytic ring, leading to stalled cup progression.
Myo1e and Myo1f play a crucial dual role in regulating adhesion and force balance during macrophage phagocytosis by coordinating spatial and temporal transitions between protrusive and contractile actin networks.
Phagocytosis requires coordinated remodeling of the actin cytoskeleton to generate protrusive and contractile forces that drive target engulfment. Class I myosins Myo1e and Myo1f (Myo1e/f) have been implicated in linking the plasma membrane to the actin network, but their specific roles during Fc-receptor (FcR)-mediated phagocytosis remain unclear. Using CRISPR-edited RAW 264.7 macrophages lacking Myo1e and Myo1f, we show that double knockout (dKO) cells exhibit markedly reduced uptake of IgG-coated beads, a phenotype that is partially rescued by re-expression of either myosin. Lattice-light-sheet and confocal imaging revealed distinct F-actin architectures corresponding to the various stages of cup progression, including basal podosome-like adhesions, individual phagocytic podosomes, or actin teeth, along the rim of the cup, and a contractile phagocytic ring formed by the reorganization of podosomes into a higher-order network. In Myo1e/f-deficient cells, podosome formation was diminished, actin teeth were largely absent, and the phagocytic ring formed prematurely, which was often accompanied by stalled cup progression and repeated engulfment attempts. Myo1e/f localized both to podosomes and to the inner surface of the phagocytic ring, non-muscle myosin II (NM2) localized to the outer surface, and the absence of Myo1e/f correlated with the diffuse distribution of NM2. In addition, Myo1e/f-deficient macrophages exhibited increased trogocytosis of antibody-opsonized HL-60 cells, indicating a shift from whole-target engulfment toward partial target ingestion. These results suggest that Myo1e/f coordinate spatial and temporal transitions between protrusive and contractile actin networks, thereby ensuring efficient phagocytic cup progression. Our findings highlight a dual role for Myo1e/f in adhesion regulation and force balance during macrophage phagocytosis.
Paul et al. (Fri,) conducted a other in Phagocytosis in macrophages. Myo1e and Myo1f double knockout vs. Wild-type or re-expression of either myosin was evaluated on Uptake of IgG-coated beads and F-actin architecture during cup progression. Double knockout of Myo1e and Myo1f in macrophages markedly reduced uptake of IgG-coated beads and caused premature formation of the phagocytic ring, leading to stalled cup progression.