FBP17 Mediates a Common Molecular Step in the Formation of Podosomes and Phagocytic Cups in Macrophages

Abstract

Macrophages act to protect the body against inflammation and infection by engaging in chemotaxis and phagocytosis. In chemotaxis, macrophages use an actin-based membrane structure, the podosome, to migrate to inflamed tissues. In phagocytosis, macrophages form another type of actin-based membrane structure, the phagocytic cup, to ingest foreign materials such as bacteria. The formation of these membrane structures is severely affected in macrophages from patients with WiskottAldrich syndrome (WAS), an X chromosome-linked immunodeficiency disorder. WAS patients lack WAS protein (WASP), suggesting that WASP is required for the formation of podosomes and phagocytic cups. Here we have demonstrated that formin-binding protein 17 (FBP17) recruits WASP, WASP-interacting protein (WIP), and dynamin-2 to the plasma membrane and that this recruitment is necessary for the formation of podosomes and phagocytic cups. The N-terminal EFC (extended FER-CIP4 homology)/F-BAR (FER-CIP4 homology and Bin-amphiphysin-Rvs) domain of FBP17 was previously shown to have membrane binding and deformation activities. Our results suggest that FBP17 facilitates membrane deformation and actin polymerization to occur simultaneously at the same membrane sites, which mediates a common molecular step in the formation of podosomes and phagocytic cups. These results provide a potential mechanism underlying the recurrent infections in WAS patients.Podosomes (see Fig. 1A) are micron-scale, dynamic, actinbased protrusions observed in motile cells such as macrophages, dendritic cells, osteoclasts, certain transformed fibroblasts, and carcinoma cells (1). Podosomes play an important role in macrophage chemotactic migration, which is critical for recruitment of leukocytes to inflamed tissues. Podosomes are both adhesion structures and the sites of extracellular matrix degradation (2). Adhesion to and degradation of the extracellular matrix are essential processes for the successful migration of macrophages in tissues. Podosomes occur in most macrophages and can be observed by differentiating human primary monocytes into macrophages with macrophage-colony stimulating factor-1 (M-CSF-1) 2 and staining the F-actin using phalloidin (3, 4). Podosomes labeled in this way appear as F-actinrich dots (see Fig. 1C). Podosome formation has recently been directly observed in vitro and in vivo in leukocyte migration through the endothelium, diapedesis (5).Phagocytosis of bacterial pathogens is one of the most important primary host defense mechanisms against infections. The phagocytic cup (see Fig. 1B) is an actin-based membrane structure formed at the plasma membrane of phagocytes, including macrophages, upon stimulation with foreign materials such as bacteria. The phagocytic cup captures and ingests foreign materials, and its formation is an essential first step in phagocytosis leading to the digestion of foreign materials (6, 7). When macrophages are stimulated by foreign materials, podosomes disappear, and phagocytic cups, which are also rich in F-actin, are formed...

Antagonistic regulation of F-BAR protein assemblies controls actin polymerization during podosome formation

“…To confirm this, we examined whether this aberrant podosomes phenotype can be rescued by further depletions of Toca proteins. Consistent with a previous study (Tsuboi et al, 2009), when FBP17 or CIP4 were knocked down, spontaneous formation of podosomes was reduced (data not shown). This effect was enhanced by simultaneous depletion of both proteins (,66% decrease; Fig.…”

“…6). This observation is somewhat surprising as the Toca protein itself has been proposed to recruit WASP to the plasma membrane (Takano et al, 2008;Tsuboi et al, 2009;Tsujita et al, 2006). We envisage that, in vivo, an initial assembly of FBP17 is triggered by WASP binding, which may induce a positive feedback loop that further propagates its assembly with WASP on the plasma membrane.…”

“…Podosomal actin structures are formed by the WASP/N-WASP-activated Arp2/3 complex at the plasma membrane (Albiges-Rizo et al, 2009;Murphy and Courtneidge, 2011). Recently, Toca proteins have been identified as key players in podosome/invadopodia formation (Chander et al, 2012;Hu et al, 2011a;Hu et al, 2011b;Linder et al, 2000;Pichot et al, 2010;Tsuboi et al, 2009), through their self-assembling property for the activation of WASP-mediated actin polymerization. However, it is unclear how the level of Toca protein assembly is determined for proper formation and dynamic turnover of podosomes.…”

Invadosomes at a glance

“…In particular, the pathway involving the Rho GTPase CDC42, WASP or N-WASP and the Arp2/3 complex, and the importance of this pathway in invadosomal F-actin polymerization, has been well documented (Linder et al, 1999;Linder et al, 2000b;Burns et al, 2001). A role for formins (which are actinnucleating proteins) has been speculated upon and is now gradually being confirmed for both podosomes [S. Blystone, personal communication; for formin-binding FBP17, see Tsuboi et al (Tsuboi et al, 2009)] and invadopodia (Lizárraga et al, 2009).…”

Spatiotemporal organization and mechanosensory function of podosomes

“…48 Also, membrane curvature-sensing proteins have been shown to be present within podosomes in macrophages. 49 How the complex interplay between membrane proteins and lipids regulates podosome texture sensing remains to be elucidated.…”