Circulation of the plasma membrane in Dictyostelium

Abstract

We have developed a fluorimetric assay with the use of the dye FM1-43 to determine the rate at which Dictyostelium amoebae endocytose their surface membrane. Our results show that they do so about once each 4-10 min. A clathrin null mutant takes its surface up only approximately 30% more slowly, showing that this membrane uptake cannot be caused by clathrin-coated vesicles. Surprisingly, Ax2 and its parent, NC4, which differ in their rates of fluid-phase internalization by approximately 60-fold, take up their surfaces at the same rates. These results show that, in axenic cells, the uptake of fluid and of surface area are separate processes. The large activity of this new endocytic cycle in both Ax2 and NC4 amoebae appears capable of delivering sufficient new surface area to advance the cells' fronts during migration.

Figure 1

FM1-43 internalization by individual Ax2 and NC4 cells. (A–F) Dye associated with Ax2 (A, C, and E) or NC4 (B, D, and F). (A and B) Surface fluorescence at 0 min after FM1-43 addition. (C and D) Cells incubated for 5 min with dye and then washed. (E and F) Same as C and D, but with an additional 25 min without dye. (G and H) Ax2 cells pretreated with azide, observed at 0 min after addition of dye (G) or after 5 min of incubation in dye and then washed (H). Differential interference contrast (DIC) images are on the left of each pair, and fluorescence images are on the right. Minor differences between pairs of DIC and fluorescence images are due to movement of the live cells. Bar, 10 μm.

Figure 2

FM1-43 uptake by Ax2 amoebae grown axenically. (A) Direct method: uptake in the absence (●) or presence of 10 mM azide (○). (B) Indirect methods: uptake at 22°C (▴) or at 0°C (×), followed by formaldehyde fixation; and uptake at 22°C, followed by solubilization in 1% Triton X-100 (▵). The lines represent the data fit to a two-exponent equation (●, ▴, ▵) or to a straight line (○, ×). In A, the uptake of FM1-43 was continued to 180 min; at this time, the level of fluorescence had nearly plateaued and the ratio of total-to-surface fluorescence was ∼2.5. A.U., arbitrary units.

Figure 3

FM1-43 uptake by Dictyostelium strains grown with bacteria and measured by the direct method. (A) NC4 (▴) and Ax2 (○). The data from Figure 2A for Ax2 cells grown axenically have been included for comparison (●). (B) mhcA⁻ (♦) and chc⁻ (▵). The lines represent the data fit to a two-exponent equation. A.U., arbitrary units.

Figure 4

FITC-dextran uptake by Ax2 and NC4 cells. Ax2 (A) and NC4 (B) cells were incubated with 2 mg/ml FITC-dextran for 5 min, washed, and fixed. Differential interference contrast images are on the left of each pair, and fluorescence images are on the right. Bar, 10 μm.