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Figure 5


Fig. 5. Effect of the Rab3A-siRNA, Rab27A-siRNA, and Rab33A-siRNA expression on the kinetics of NPY-Venus release. (A) Typical sequential images of a single NPY-Venus vesicle observed after high-KCl (70 mM) stimulation of cells expressing control vector (control), Rab3A-siRNA (Rab3A siRNA), Rab27A-siRNA (Rab27A siRNA) or Rab33A-siRNA (Rab33A siRNA), acquired at 200-millisecond intervals, through a TIRF microscope. The third column of images (0.8 seconds) shows a diffuse cloud of NPY-Venus fluorescence, and the fourth column of images (1.2 second) shows disappearance of the spot. (B) Time course of the fluorescence changes measured in the center of NPY-Venus vesicles in the control ({blacksquare}), Rab3A-siRNA-expressing ({circ}), Rab27A-siRNA-expressing ({triangleup}), and Rab33A-siRNA-expressing ({triangledown}) cells. Mean fluorescence intensity before fusion was set as 100% (n=15 vesicles in each experiment). Bar, 2 µm. Images were acquired every 200 milliseconds under each set of conditions. Note that silencing Rab3A, Rab27A, or Rab33A had no effect on the kinetics of vesicle fusion of the dense-core vesicles.