Figure 4.

Characterization of lysosomal exocytosis by monitoring the release of a lumenal marker. Lysosomes in NRK cells were loaded with FITC dextran and cells were treated with 10 μm A23187 calcium-ionophore. Although the epifluorescence image (A) predominantly demonstrates the perinuclear population of lysosomes, TIR-FM (B) allows identification of lysosomes near the plasma membrane. In response to the addition of calcium ionophore there is a reduction in lysosomal movement and ∼7% of the total lysosomes in the evanescent field exocytose (Video 3). (C) Most of the exocytosing lysosomes are predocked (arrowhead), whereas a few recent arrivals (arrow) also exocytose. Time stamp indicates the time elapsed since the addition of ionophore. (D) Quantification of changes in the total intensity and (half-width)² of the fluorescence of exocytosing dextran. X-axis represents the time starting from when the lysosome ceased moving before initiation of fusion. Note that the spread phase for dextran (a lumenal marker) is much shorter compared with the spread phase for a CD63-EGFP (a membrane marker; Fig. 3 C).