An Essential Role for Talin during α_Mβ₂-mediated Phagocytosis

Abstract

The cytoskeletal, actin-binding protein talin has been previously implicated in phagocytosis in Dictyostelium discoideum and mammalian phagocytes. However, its mechanism of action during internalization is not understood. Our data confirm that endogenous talin can occasionally be found at phagosomes forming around IgG-and C3bi-opsonized red blood cells in macrophages. Remarkably, talin knockdown specifically abrogates uptake through complement receptor 3 (CR3, CD11b/CD18, ␣ M ␤ 2 integrin) and not through the Fc ␥ receptor. We show that talin physically interacts with CR3/␣ M ␤ 2 and that this interaction involves the talin head domain and residues W747 and F754 in the ␤ 2 integrin cytoplasmic domain. The CR3/␣ M ␤ 2 -talin head interaction controls not only talin recruitment to forming phagosomes but also CR3/␣ M ␤ 2 binding activity, both in macrophages and transfected fibroblasts. However, the talin head domain alone cannot support phagocytosis. Our results establish for the first time at least two distinct roles for talin during CR3/␣ M ␤ 2 -mediated phagocytosis, most noticeably activation of the CR3/␣ M ␤ 2 receptor and phagocytic uptake. INTRODUCTIONPhagocytosis is an essential physiological function, common to most eukaryotic cell types. From serving a feeding role in amoebae, phagocytosis is observed in Metazoa as a homeostatic process that ensures the removal of microorganisms and apoptotic cells (Desjardins et al., 2005). Classically, phagocytosis is a multistep process that sequentially involves receptor-mediated particle recognition, actin-driven uptake, phagosome maturation and particle clearance. Numerous phagocytic receptors exist that can bind their target directly or indirectly through opsonins (Underhill and Ozinsky, 2002). Receptors for phagocytosis can show constitutive or inducible binding activities, as illustrated for the two bestcharacterized phagocytic receptors: the Fc ␥ receptor (Fc␥R) for complexed IgG and complement receptor 3 (CR3, CD11b/ CD18, ␣ M ␤ 2 integrin), respectively (Bianco et al., 1975). Ligand-bound receptors classically zipper around the phagocytic prey and induce intracellular signaling cascades that lead to the activation and recruitment of signaling and adaptor molecules at sites of particle binding. These locally assembled signaling complexes reorganize the actin cytoskeleton and regulate membrane dynamics underneath bound particles through the activation of Rho-and Arf-family GTPbinding proteins, respectively (Cougoule et al., 2004). According to the zipper model, phagocytosis of bound particles requires continual ligation of phagocytic receptors around the whole phagocytic object, at least for spherical particles Champion and Mitragotri, 2006). Several cytoskeletal proteins have been shown to be recruited to phagocytic cups, although their role is not always defined. Talin, a cytoskeletal protein of 2541 amino acids and 270 kDa has been repeatedly implicated in phagocytosis. Immunofluorescence studies of phagocytozing macrophages have shown that talin accumulates...

RhoG is required for both FcγR- and CR3-mediated phagocytosis

“…1D-F), talin RNAi induced concomitant decreases in the association and phagocytic indices (Fig. 1D,E), consistent with its crucial role in CR3 activation (Lim et al, 2007). RhoA RNAi, but not RNAi against RhoB or RhoC, strongly reduced the CR3-mediated percentage of phagocytosis (,50% decrease, Fig.…”

“…1A-C) (Lim et al, 2007). The largest reduction in FccR percentage phagocytosis was produced by RhoG RNAi (,50%, Fig.…”

Talin at a glance

“…Talin (but not vinculin) is also required for both F-actin and integrin accumulation at the immunological synapse (Nolz et al, 2007), and for the chemokine-induced increase in affinity of integrin VLA-4 (α4β1) for VCAM1, which is important in lymphocyte-endothelial cell adhesion (Manevich et al, 2007). In macrophages, talin 1 is required for phagocytosis that is mediated by αMβ2 integrin (complement receptor 3) (Lim et al, 2007), whereas a talin-PIPK1γ90 complex has been shown to play a novel role in clathrin-mediated endocytosis in the neuronal synapse (Morgan et al, 2004).…”

Cathepsin X cleaves the β2 cytoplasmic tail of LFA‐1 inducing the intermediate affinity form of LFA‐1 and α‐actinin‐1 binding

“…On the other hand, the association of the cytoplasmic b 2 tail with actin-binding proteins results in modification of the actin cytoskeleton and, hence, cell migration. Different consensus sequences of the b 2 cytoplasmic tail are known to associate with the actin-binding proteins talin [9], filamin [10], radixin [11] and a-actinin-1 [12], and also with the signaling proteins FAK [13], cytohesin 1 [14] and cytohesin 3 [15] that are the final effectors of inside-out signaling pathways.…”