Myosin-II activity at the phagocytic synapse with opsonized RBC targets is inhibited by CD47 and enhanced by target rigidity. (A) Heat map summary and hierarchical clustering of normalized results for the various experiments. The dendrogram indicates that the macrophage response to rigid GA discocytes is distinct from that of native RBC targets and that blocking CD47 on deformable RBCs (but not on GA discocytes) reduces the difference, as does blebbistatin pretreatment of macrophages encountering GA discocytes (CD47⁺ GA-Disc +Blebb). CD47 inhibition is partially rescued with rigid but rounded GA stomatocytes (CD47⁺ GA-Stom), unless CD47 blocked (CD47⁻ GA-Stom). (B) Macrophage recognition of IgG opsonin on the surface of RBCs by FcγR phagocytic receptors activates cytoskeletal proteins including myosin-II. Effective signaling via SIRPA to phosphatases does occur with CD47 on either flexible self-cells or sufficiently rounded but rigid self-cells. RBC rigidity has the effect of rapidly and strongly activating adhesion and myosin-II contractions. Macrophages pinch deformable CD47-blocked native RBCs at their midpoint and then fully engulf these RBCs in a manner analogous to a deflated balloon pulled through a napkin ring (inset schematic). Rigid discocytes maintain their shape throughout engulfment, which limits contact between macrophage receptors and ligands, particularly SIRPA and CD47 on the RBCs, and thus the spacious phagosome may act as a signaling barrier. Rounded GA stomatocytes show tightly apposed macrophage pseudopods and rescued CD47 signaling. (C) Schematic of myosin-II assembly in phagocytosis in which myosin-II dimers assemble and contract actin filaments in response to rigidity of human RBCs while becoming more disorganized in response to CD47-SIRPA-mediated activation of immunoinhibitory phosphatase, SHP-1. We showed previously⁷  that phosphorylation of tyrosines in myosin-IIA’s head and tail activate the motor, increasing its accumulation at the phagocytic synapse and increasing the efficiency of phagocytic uptake, whereas CD47-SIRPA’s activation of the tyrosine phosphatase SHP-1 deactivates myosin-IIA. Blebbistatin blocks the activity of myosin II ATPase and thus the ability of the head to generate contractile forces, relaxing the assembled actomyosin fibers. The SHP-1 inhibitor NSC87877 reduces inhibition by CD47 and thus increases RBC uptake.