Stefan Walbaum's research while affiliated with Max Planck Institute of Molecular Cell Biology and Genetics and other places

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and knockout mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80⁺ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ-chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcγRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ-chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and knockout mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ-chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcgRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ-chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.

G protein–coupled receptor (GPCR) signaling is required for the navigation of immune cells along chemoattractant gradients. However, chemoattractant receptors may couple to more than one type of heterotrimeric G protein, each of which consists of a Gα, Gβ, and Gγ subunit, making it difficult to delineate the critical signaling pathways. Here we used knockout mouse models and time-lapse microscopy to elucidate Gα and Gβ subunits contributing to complement C5a receptor–mediated chemotaxis. Complement C5a–mediated chemokinesis and chemotaxis were almost completely abolished in macrophages lacking Gnai2 (encoding Gαi 2 ), consistent with a reduced leukocyte recruitment previously observed in Gnai2 -/- mice, whereas cells lacking Gnai3 (Gαi 3 ) exhibited only a slight decrease in cell velocity. Surprisingly, C5a-induced Ca ²⁺ transients and lamellipodial membrane spreading were persistent in Gnai2 -/- macrophages. Macrophages lacking both Gnaq (Gα q ) and Gna11 (Gα 11 ) or both Gna12 (Gα 12 ) and Gna13 (Gα 13 ) had essentially normal chemotaxis, Ca ²⁺ signaling, and cell spreading, except Gna12/Gna13-deficient macrophages had increased cell velocity and elongated trailing ends. Moreover, Gnaq / Gna11 -deficient cells did not respond to purinergic receptor P2Y 2 stimulation. Genetic deletion of Gna15 (Gα 15 ) virtually abolished C5a-induced Ca ²⁺ transients, but chemotaxis and cell spreading were preserved. Homozygous Gnb1 (Gβ 1 ) deletion was lethal, but mice lacking Gnb2 (Gβ 2 ) were viable. Gnb2 -/- macrophages exhibited robust Ca ²⁺ transients and cell spreading, albeit decreased cell velocity and impaired chemotaxis. In summary, complement C5a–mediated chemotaxis requires Gαi 2 and Gβ 2 , but not Ca ²⁺ signaling, and membrane protrusive activity is promoted by G proteins that deplete phosphatidylinositol 4,5-bisphosphate.