Fig. 3. Molecular characterization of the phagosomal-induced actin polymerization. (A) Polymerization is inhibited by low cyto. D concentrations. Two hour phagosomes were incubated in polymerization conditions, without (control) or with 100 nM cyto. D. The number of positive phagosomes was determined by fluorescence microscopy. The errors reported are the SD from three separate experiments. (B) Endogenous actin filaments are not required for the phagosomal-induced actin assembly. F–actin beads or 2 h phagosomes were pre-incubated in buffer F for 10 min at 25°C, with (hatched bars) or without (black bars) 2 μM cyto. D, and purified by flotation in PHEM buffer with protease inhibitors. Polymerization of actin was determined in the absence (F–actin beads) or the presence (2 h phagosomes) of Tβ4 using the flow cytometry assay. The errors reported are the SD from three separate experiments. (C) Labeling of assembled filaments with myosin S–1 visualized by electron microscopy, showing the presence of actin bundles emanating from the membrane fragments attached to a latex bead (arrowhead) (left). Right panel, higher magnification of insert; arrowheads symbolize the direction of polarity. Bars, 0.5 μm. (D) Actin assembly on the surface of phagosomes. Polylysine beads incubated first with fluorescein–G–actin (Actin 1) and subsequently with rhodamine–G–actin (Actin 2) are shown in the upper panel. Two hour phagosomes incubated first with rhodamine–G–actin–Tβ4 (Actin 1) and subsequently with fluorescein–G–actin–12 μM Tβ4 (Actin 2) are shown in the lower panel. Optical sections of different representative fields are shown. Fluorescein–actin (green), rhodamine–actin (red) and latex beads (visualized by phase contrast, here in dark blue) images are overlapped. Bar, 5 μm.