Figure 3.

Lysosomal membrane and lumenal markers confirm that calcium induces exocytosis of lysosomes. NRK cells labeled with either CD63-EGFP (A–C) or CD63-EGFP (D) and 10 kD Texas red–dextran were treated with 10 μm A23187 ionophore and imaged using TIR-FM. A region from A that shows a cell with CD63-EGFP–labeled lysosomes in the evanescent field. A lysosome that underwent exocytosis has been enlarged in B; the arrow indicates a CD63-EGFP–labeled lysosome that moved into the evanescent field during the period of observation and fused to the plasma membrane. Note that it takes ∼3 s for the fluorescence to diffuse out along the membrane. (C) The quantification of the peak, total intensity and (half-width)² of the fluorescence for a lysosome that undergoes fusion (the values for peak intensity has been multiplied by a factor of five). The point when lysosome ceased moving before initiation of fusion was used as t = 0. (D) Representative images of a lysosome double labeled for membrane (green; using CD63-EGFP) and lumen (red; Texas red dextran) that underwent calcium-induced fusion. The arrow points to a lysosome that underwent exocytosis. The time stamp represents the time elapsed after the addition of ionophore. Note that the dextran fluorescene (red) diffuses much faster compared with the diffusion of the fluorescence of the membrane marker (green; Video 2).