Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion

Abstract

Edited by Velia M. FowlerMacrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial (Escherichia coli) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan (Saccharomyces cerevisiae) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading.Phagocytosis is a specialized form of endocytosis, which requires localized actin polymerization to engulf large particles (Ͼ ϳ0.5 m), and initially involves particle binding to phagocytic receptors, which triggers phagocytic cup formation through the activation of kinases, such as Syk, and Rho GTPases, such as the Cdc42 and Rac subfamilies (1). Although phagocytosis has been investigated in-depth, the involvement of filopodia (finger-like projections containing bundled actin filaments (2)) in actually capturing and clearing particles has not been conclusively demonstrated, except for the historical observations of Metschnikoff (3) and more recent work using brightfield microscopy (4 -7).Young et al. (4) observed using time-lapse differential interference contrast (DIC) 2 imaging that Escherichia coli expressing invasin, a transmembrane protein of Yersinia pseudotuberculosis, could enter Hep-2 (HeLa-derived) cells via filopodia. Using another approach, coating of magnetic microbeads with invasin, Vonna et al. (5) found that the adhesion of beads to filopodial tips induced pulling toward the cell body. Similarly, Kress et al. (6) reported that filopodia act as "phagocytic tentacles" and pulled IgG-coated beads in an optical trap with discrete steps, suggesting that a motor pro...

Roles for Ena/VASP proteins in FMNL3-mediated filopodial assembly

“…These structures serve multiple purposes, including cell migration, cell-substratum adhesion and cell-cell adhesion (Blanchoin et al, 2014;Chhabra and Higgs, 2007;Faix et al, 2009;Yang and Svitkina, 2011). In addition, filopodia are often the initial sites of microbial contact during phagocytosis or viral infection (Chang et al, 2016;Horsthemke et al, 2017). Both the length and the dynamics of filopodia can vary widely depending on cell type and context.…”

“…In addition to these two models, myosin-X (symbol MYO10) has been shown to be involved in filopodial assembly in multiple cell lines. Overexpression of myosin-X leads to increased filopodial assembly (Berg and Cheney, 2002;Bohil et al, 2006;Dent et al, 2007;Tokuo et al, 2007), whereas myosin-X suppression or deletion leads to a decrease in filopodia number (Heimsath et al, 2017;Horsthemke et al, 2017;Tokuo et al, 2007). The mechanism by which myosin-X promotes filopodial assembly is unclear.…”

Time-lapse Imaging of Mouse Macrophage Chemotaxis

“…Using this system and time-lapse, phase-contrast microscopy, we developed an assay to image mouse resident peritoneal macrophages migrating in a chemotactic complement C5a gradient 31,34,35,36 . This assay, combined with knockout mouse models, proved instrumental in the investigation of the roles of various Rho GTPases and motor proteins in macrophage morphology, motility, and chemotaxis 31,34,35,36,37 . We have also used this approach to image human peripheral blood monocytes migrating on a 2D surface or in a 3D collagen type I matrix…”

Tunneling Nanotubes and the Eye: Intercellular Communication and Implications for Ocular Health and Disease

“…Overexpression of Myo10 in a variety of cell types increases the number of filopodia emanating from the cell surface, while Myo10 knockdown reduces filopodia number [85,86,91,99,100]. Overexpression of Myo10, but not VASP or fascin, also induced TNT formation [87].…”