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First published online 20 November 2002
doi: 10.1242/jcs.00235

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Phosphoinositide-3-kinase-independent contractile activities associated with Fc-receptor-mediated phagocytosis and macropinocytosis in macrophages

¹ Department of Histology and Cell Biology, Kagawa Medical University, Miki, Kagawa 761-0793, Japan
² Department of Biochemistry, Kagawa Medical University, Miki, Kagawa 761-0793, Japan
³ Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA

View larger version (126K): [in a new window]   Fig. 4. Time-lapse sequences of living RAW264.1 macrophages transfected with pEGFP-actin, showing actin dynamics during phagocytosis of IgG-Es. (A) In a control cell, IgG-Es induced F-actin assembly where they were bound, resulting in phagocytic-cup formation. It was noteworthy that the IgG-Es was deformed during phagocytic-cup formation, suggesting that the F-actin ring constricted the phagocytic cup and squeezed the IgG-Es. Subsequently, the F-actin ring seemed to close at the top of the cup. F-actin disappeared immediately after phagocytic cups closed into intracellular phagosomes. (B) In a ML-7-treated cell, F-actin assembly and phagocytic-cup extension occurred, but the process was slower and the cup was shallower than in control. Even though phagocytic cups were formed, they did not constrict or close. The time interval between frames is 10 seconds. Bars, 10 µm. The supplemental video materials are available in the online version [Video 1 (http://www.jcs.biologists.org/supplemental)].

View larger version (113K): [in a new window]   Fig. 7. Time-lapse sequences of ruffling and macropinocytosis. Macrophages were stimulated with 2000 U ml-1 M-CSF and treated with 0.1% dimethyl sulfoxide (control; A), 100 nM wortmannin (B), 10 µM ML-7 (C) or 10 µM cytochalasin D (D). Numbers indicate time sequence with 10-second intervals. Ruffles were seen as phase-dense bands in the periphery of the cells. (A) In control cells, phase-bright macropinosomes were formed by closing circular ruffles (arrows). (B) Wortmannin did not inhibit circular ruffle formation but did inhibit the closure of circular ruffles into macropinosomes (arrows). (C) In ML-7, ruffle movement was markedly reduced. No circular ruffle formation was observed. Only simple centripetal movement of ruffles was seen. (D) Cytochalasin D stopped the ruffle movement completely. Bar, 10 µm. The supplemental video materials are available in the online version [Video 2 (http://www.jcs.biologists.org/supplemental)].

View larger version (20K): [in a new window]   Fig. 1. Effects of various inhibitors for PI3K, MLCK and F-actin on binding and phagocytosis of IgG-Es. Macrophages were fed IgG-Es in RB medium containing 0.1% dimethyl sulfoxide (Control), 50 µM LY294002 (LY294), 100 nM wortmannin (Wort), 10 µM ML-7, 20 µM ML-7 or 10 µM cytochalasin D (CyD). After 30-minute incubation at 4°C (for binding; white bars) or 37°C (for phagocytosis; black bars), the binding/phagocytic index (IgG-Es per cell) was scored as described in Materials and Methods. Bars show the mean and standard deviation of triplicate determinations (n=3) in a single experiment. Data are representative of at least three independent experiments.

View larger version (76K): [in a new window]   Fig. 2. Phase-contrast and fluorescence microscopy of IgG-Es and F-actin during phagocytosis in macrophages treated with 0.1% dimethyl sulfoxide (control), 100 nM wortmannin or 10 µM ML-7. Macrophages were pretreated with one of the drugs for 15 minutes, fed IgG-Es and incubated for 30 minutes in the presence of the drug. After fixation, extracellularly exposed IgG-Es and F-actin were stained with FITC—anti-rabbit-IgG and rhodamine phalloidin, respectively. In control cells (A-C), IgG-Es were internalized in phagosomes and/or phagolysosomes, and were scarcely associated with F-actin. In wortmannin-treated cells (D-F), IgG-Es seemed to be surrounded by F-actin-rich protrusions but remained partially exposed to extracellular fluid. ML-7-treated macrophages (G-I) appeared similar to wortmannin-treated cells. (A,D,G) Phase-contrast images. (D,E,H) FITC images showing extracellular IgG-Es. (C,F,I) Rhodamine images showing F-actin. Bars, 10 µm.

View larger version (117K): [in a new window]   Fig. 3. SEM of IgG-E phagocytosis in macrophages treated with the drugs. (A) A phagocytic cup in a control macrophage after 7-minute incubation with IgG-Es. The phagocytic cup is conformed closely to the surface of IgG-Es. The cylindrical cup appeared to have constricted the IgG-Es. After 30 minutes, IgG-Es were no longer seen on the cell surface, because all had been internalized by the cells (not shown). (B) In wortmannin-treated macrophages, many phagocytic cups were observed on the dorsal surface of macrophages. The configuration of phagocytic cups was similar to that in control cells at 7 minutes after phagocytosis. (C) ML-7 treatment also did not inhibit phagocytic-cup formation. Most strikingly, phagocytic cups were not closely apposed to the surfaces of IgG-Es. (D) Cytochalasin D inhibited phagocytic-cup formation. IgG-Es bound to the plane dorsal surface of the cells. Electron micrographs were pseudocolored using Adobe Photoshop. Bars, 5 µm.

View larger version (115K): [in a new window]   Fig. 5. Immunolocalization of myosin II and phosphorylated myosin light chain (P-MLC) in ruffles and phagocytic cups of macrophages. Immunofluorescence of myosin II (A) and P-MLC (B) in macrophages stimulated with M-CSF showed that MLC of myosin II are predominantly phosphorylated in cell-surface ruffles. (C,D) In control macrophages phagocytosing IgG-Es (7 minutes after feeding with IgG-Es), P-MLC localized in some phagocytic cups (arrows) but not in phagosomes (open arrows). (E,F) ML-7 treatment greatly reduced the amount of P-MLC in phagocytic cups. Phase-contrast images C and E correspond to fluorescence images D and F, respectively. Bars, 10 µm.

View larger version (14K): [in a new window]   Fig. 6. Fluorometric quantitation of macropinocytosis. Macrophages were pretreated with 0.1% dimethyl sulfoxide (Control), 50 µM LY294002 (LY294), 100 nM wortmannin (Wort), 10 µM ML-7 or 10 µM cytochalasin D (CyD) for 15 minutes, and incubated with 1.0 mg ml-1 FDx150 in RB medium for 30 minutes to allow macropinocytosis. The fluorescence of cell lysates was assayed by spectrofluorometry. The values represent the mean plus or minus the standard deviation of triplicate determinations. Similar results were obtained in two additional experiments.

View larger version (137K): [in a new window]   Fig. 8. Digital image analysis of ruffling activities in control (A), wortmannintreated (B), ML-7-treated (C) and cytochalasin-D-treated cells (D). Subtracted images (right) were obtained by subtracting the images (center) from the frames taken 20 seconds earlier (left), then adding 120 to the gray value of each pixel. In subtracted images, the standard deviations (SD) of gray values of the cell area are shown. Bar, 10 µm.