Figure 8.

The role of dynamin A in pinocytosis and phagocytosis. For quantification of fluid-phase marker uptake, cells were incubated with 1 mg/ml FITC-dextran, and intracellular fluorescence was measured at the time points indicated. The amount of FITC-dextran internalized was plotted as percent of total uptake of Ax2 cells after 160 min. (A) In comparison to wild-type Ax2 cells (closed circles) and a rescued cell line (open circles), the extent to which the marker was accumulated was approximately twofold reduced in dymA⁻ cells (open triangles). The initial rate of uptake for the first 40 min was 33% slower for dymA⁻ than the parental wild-type or rescue cells. (B) For analysis of exocytosis, cells were incubated with 1 mg/ml FITC-dextran for 2 h, washed, and incubated with fresh medium for the time points indicated. Intracellular fluorescence was measured and plotted as percent of FITC-dextran remaining in the cells. Similar rates of exocytosis were determined for dymA⁻ cells and cells producing dynamin A. (C) Intracellular transit time of fluid phase was measured by incubating the cells in 5 mg/ml FITC-dextran for 10 min and transfer to fresh media for the time indicated. Intracellular fluorescence was measured and plotted as percent of FITC-dextran remaining in the cells. In dymA⁻ cells transit time was prolonged more than twofold. (D) Phagocytotic ability was assayed by following the uptake of 1 μm polystyrene microspheres (100 beads/cell) in axenic medium. In dymA⁻ cells phagocytosis is increased by 50% compared with control cells. All the assay data are normalized to protein content and represent averages from at least three independent experiments.