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Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

University of Massachusetts Medical School, Department of Physiology, 377 Plantation, Room 327, Worcester, MA 01605, USA

View larger version (24K): [in a new window]   Fig. 1. Similar opsonization of hard and soft polyacrylamide microbeads and sheets. (a) Average fluorescent intensities of opsonized beads labeled with red fluorescent secondary antibodies. Intensities were measured with 100 each of stiff and soft beads from three preparations. Error bars indicate the standard deviation between each experiment. (b,c) Images of stiff (b) or soft (c) polyacrylamide surfaces coated with anti-BSA IgG and labeled with fluorescent carboxylate beads coated with anti-rabbit antibodies. The average intensities were within 7% of each other for the beads and 15% for the sheets.

View larger version (69K): [in a new window]   Fig. 2. Detection of ingested polyacrylamide beads. Macrophages were fed with stiff, opsonized polyacrylamide beads with fluorescent dextran trapped inside. Unlike beads that stuck to the side of the cell (arrowhead), internalized beads are not readily visible with phase optics (a; long arrow). However, a combination of phase and fluorescence optics easily reveals all the beads (b; arrow and arrowheads). (c) Stereo reconstruction of the same cell stained with rhodamine phalloidin, showing a closed phagocytic cup around the internalized bead (long arrow). A patch of actin is concentrated beneath the external bead (arrowhead). Bar, 4 µm.

View larger version (50K): [in a new window]   Fig. 3. Efficiency of phagocytosis of stiff or soft objects during Fc-receptor-mediated phagocytosis. (a) Stiff polyacrylamide beads (left two bars) are six times more likely to be phagocytosed than soft beads (right two bars). Beads without antibody coating are poorly phagocytosed. Phagocytic index is defined as the average number of beads internalized per cell. These data were compiled from six independent experiments with 100 cells counted in each experiment. (b) Frustrated phagocytosis on progressively softer opsonized polyacrylamide sheets. Data were compiled from three independent experiments. Stiff sheets cause sixfold more cells to undergo frustrated phagocytosis than do soft sheets. Error bars indicate the standard deviation between experiments. Panels c-e show images of macrophages phagocytosing opsonized stiff beads (c-d) and sheets (e). Internalized beads can be easily discriminated from beads that are merely attached to the cell surface based on fluorescence and phase images. Panels f-h show cells that failed to phagocytose opsonized soft beads (f-g) or sheets (h). Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 4. Organization of actin filaments, paxillin and phosphotyrosine following the binding of polyacrylamide beads. Actin (a), phosphotyrosine (c), and paxillin (e) are concentrated beneath bound stiff beads (arrows). However, only phosphotyrosine (d) and paxillin (f), but not actin (b), are concentrated beneath bound soft beads (b,d,f; arrows). Beads are represented as gray circles in panels a and b. Owing to the high intensity of actin staining, weak fluorescence of these beads is not detectable in these prints. Bar, 6 µm.

View larger version (50K): [in a new window]   Fig. 5. Effects of constitutively active Rho GTPases on the phagocytosis of soft particles. (a) Microinjection of L61Rac1, but not L63RhoA, stimulates the uptake of soft beads. Protein solutions were microinjected into 230 (L61Rac1), 120 (L63RhoA) or 100 cells (buffer control), before the presentation of opsonized soft beads. (b) Rhodamine phalloidin staining of serum-starved REF cells microinjected with L63RhoA reveals strong staining of stress fibers compared with surrounding uninjected cells, confirming the activity of the injected protein. Error bars indicate the standard deviation between three independent experiments. Bar, 10 µm.

View larger version (50K): [in a new window]   Fig. 7. Inhibition of LPA-stimulated phagocytosis of soft particles by dominant negative Rac1. (a) Macrophages microinjected with N17Rac1 or buffer were fed with opsonized hard or soft beads, with or without the additional incubation with LPA. The phagocytic index for each condition was determined with a total of 70-100 cells in three independent experiments. (b) Macrophages microinjected with C3 transferase or buffer were fed with opsonized beads, with or without the additional incubation with LPA. The phagocytic index for each condition was determined with a total of 120-150 cells in three independent experiments. Error bars indicate the standard deviation between experiments. Efficacy of the C3 transferase was confirmed by the induction of cell retraction upon microinjection into serum-starved REF-52 cells. Bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 6. Stimulation of phagocytosis of soft particles by LPA. (a) Opsonized beads were incubated with 3 µM LPA before presenting to murine macrophages and the phagocytic index determined as in Fig. 1. The data were compiled from four independent experiments, error bars indicate the standard deviation between experiments. LPA-stimulated the ingestion of opsonized soft beads, but not soft beads labeled with BSA alone. LPA also induced a concentration of actin filaments beneath soft beads (c, arrows), when compared with the control (b, arrows). Bar, 3 µm.

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