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...