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. 2008 Nov;19(11):4628–4639. doi: 10.1091/mbc.E08-02-0223

Figure 5.

Figure 5.

MTs and kinesin are essential for ruffle formation and C3bi-target binding. (A) Immunostaining of MTs and actin in control or PMA- or LPS-stimulated RAW264.7 cells. After C3bi-sRBC binding, cells were fixed and immunostained for α-tubulin (red) and actin (green). Arrows indicate MTs at the site of actin-rich membrane ruffles associated with C3bi-sRBCs. DIC image is shown in insets where C3bi-sRBCs are indicated by arrowheads. (B) Cytoskeletal requirements for ruffle formation and C3bi-sRBC binding was determined by treating cells with nocodazole (Noc) or cytochalasin D (CD) during PMA activation before C3bi-particle binding and SEM processing. Noc treatment inhibited both membrane ruffle formation and particle binding, whereas in Noc washout (Noc WO) experiments, binding of C3bi-sRBCs through membrane ruffles was observed (arrows). Taxol stimulation of macrophages induced ruffles associated with C3bi-sRBCs. Scale bars, 10 μm. (C) Graphic representation of total number of C3bi-sRBCs bound per 100 macrophages and number of bound C3bi-sRBCs associated with membrane ruffles in each treatment (see Materials and Methods). Data represents mean and SEM from three separate experiments. * p < 0.05 compared with control, untreated macrophages. (D) Cartoon illustration of TIRF microscopy visualization of the actin and MT cytoskeletons during Mac-1–mediated frustrated phagocytosis. (E) RAW264.7 cells serum starved in suspension for 3 h were pretreated with LPS and plated on C3bi-coated coverslips for 25 min. Cells were fixed and immunostained for CLIP-170 (red) and actin (green) and imaged using TIRF microscopy. CLIP-170, which decorates MT plus ends, was observed within peripheral actin-rich protrusions at the cortical regions engaged in phagocytosis (arrows). Scale bar, 10 μm. (F) Quantification of Mac-1–mediated frustrated phagocytosis where the number of RAW264.7 cells adhered to the C3bi-coated coverslips were counted for each treatment. * p < 0.05 compared with control, untreated cells. Data are mean and SEM from three separate experiments.