Evidence for a molecular complex consisting of Fyb/SLAP, SLP-76, Nck, VASP and WASP that links the actin cytoskeleton to Fcγ receptor signalling during phagocytosis

Abstract

Phagocytosis by macrophages and neutrophils involves the spatial and temporal reorganisation of the actin-based cytoskeleton at sites of particle ingestion. Local polymerisation of actin filaments supports the protrusion of pseudopodia that eventually engulf the particle. Here we have investigated in detail the cytoskeletal events initiated upon engagement of Fc receptors in macrophages. Ena/vasodilator-stimulated phosphoprotein (VASP) proteins were recruited to phagosomes forming around opsonised particles in both primary and immortalised macrophages. Not only did the localisation of Ena/VASP proteins coincide, spatially and temporally, with the phagocytosis-induced reorganisation of actin filaments, but their recruitment to the phagocytic cup was required for the remodelling of the actin cytoskeleton, extension of pseudopodia and efficient particle internalisation. We also report that SLP-76, Vav and profilin were recruited to forming phagosomes. Upon induction of phagocytosis, a large molecular complex, consisting in part of Ena/VASP proteins, the Fyn-binding/SLP-76-associated protein (Fyb/SLAP), Src-homology-2 (SH2)-domain-containing leukocyte protein of 76 kDa (SLP-76), Nck, and the Wiskott-Aldrich syndrome protein (WASP), was formed. Our findings suggest that activation of Fcγ receptors triggers two signalling events during phagocytosis: one through Fyb/SLAP that leads to recruitment of VASP and profilin; and another through Nck that promotes the recruitment of WASP. These converge to regulate actin polymerisation, controlling the assembly of actin structures that are essential for the process of phagocytosis.

PtdIns(3,4)P2, Lamellipodin, and VASP coordinate cytoskeletal remodeling during phagocytic cup formation in macrophages

“…Lpd is central to the regulation of cytoskeletal dynamics 33 as it interacts with multiple actin regulators such as the Ena/VASP family proteins 32,36,65 and the SCAR/WAVE regulatory complex 36,60 . Ena/VASP proteins localize to the phagocytic cup and are indispensable for phagocytosis: Coppolino et al 43 reported that upon binding of Ena/VASP to a cytosolic GFP-ActA construct, phagocytosis was inhibited. In the current study, we were able to validate these findings using a more thorough method that entailed mistargeting Ena/VASP proteins to the mitochondria.…”

Recruitment of the adaptor protein Nck to PECAM-1 couples oxidative stress to canonical NF-κB signaling and inflammation

“…Interaction between Nck and the T cell receptor (TCR) stimulates PAKdependent actin polymerization, which is important for the formation of the immunological synapse (31,32), and mice deficient in both Nck1 and Nck2 show reduced thymic selection and reduced TCR sensitivity (33,34). Similarly, Nck modulates actin remodeling during phagocytosis in neutrophils and macrophages (35). Work from our group shows an important role for Nck in mediating endothelial activation (27); however, the mechanisms of action appear to be independent of actin remodeling responses.…”

A Noncanonical Binding Site in the EVH1 Domain of Vasodilator-Stimulated Phosphoprotein Regulates Its Interactions with the Proline Rich Region of Zyxin

“…The processive actin polymerase VASP (vasodilator-stimulated phosphoprotein) is an important regulator of the organization of the actin cytoskeleton. − VASP and its homologues, Mena and Evl, are involved in the formation of lamellipodia and filopodia, , as well as the cellular processes of phagocytosis, cell migration, ,, and neuron axon guidance . VASP functions both as an actin polymerase and as an anti-capping protein, ,, by protecting the barbed end of actin filaments from capping proteins.…”