Myo1e/f regulate phagocytic podosomes to promote efficient cup closure in macrophages

Phagocytosis requires coordinated remodeling of the actin cytoskeleton to drive target engulfment. Class I myosins Myo1e and Myo1f contribute to Fc-receptor-mediated phagocytosis. Using CRISPR-edited RAW 264.7 macrophages, we show that cells lacking Myo1e/f exhibit reduced uptake of IgG-coated beads. 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 actin nodes along the rim of the cup, and a highly contractile, actin-rich phagocytic ring. In Myo1e/f-deficient cells, podosome formation was diminished, and the phagocytic ring formed prematurely, which was often accompanied by the strong deformation of the phagocytic targets and repeated engulfment attempts. Myo1e/f localized both to podosome-like structures 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 broader distribution of NM2. In addition, Myo1e/f-deficient macrophages exhibited increased trogocytosis of antibody-opsonized HL-60 cells. 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.