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Figure 4


Fig. 4. TIRF analysis of ANF-EGFP secretion in wild-type cells and cells lacking Syt I and Syt IX (Syt I/IX-null cells). (A) Representative footprints of wild-type and Syt I/IX-null cells. TIRF images were acquired of live cells resting in basal medium. Square insets are 1 µm2. (B) DCVs in each footprint were counted and divided by the total footprint area. The frequency distribution of DCVs for wild-type and Syt I/IX-null cells was similar. The average number of DCVs per µm2 (± s.d.) was 7.4±1.5 for wild-type and 7.5±1.1 for Syt I/IX-null cells (n=30). (C) Single ANF-EGFP secretion events are shown as sequential images at 4 Hz (bottom) with fluorescence intensity plots (top). The event shown on the left corresponds to full fusion with release of ANF-EGFP whereas the event on the right corresponds to transient fusion event with incomplete ANF-EGFP release; x=2.5 seconds and y=10 arbitrary units. (D) Wild-type and Syt I/IX-null cells expressing ANF-EGFP were stimulated with depolarization medium and images were acquired at 0.25-second intervals. Fusion events were counted manually as a flash (or puff) of fluorescence in wild-type cells or Syt I/IX-null cells and the sum of fusion events in 10-second intervals was plotted (n=21). The number of fusion events was drastically reduced in the Syt I/IX-null cells. (E) Same data as in C plotted as the sum of the average number of fusion events per cell over time (mean ± s.d.).





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