Figure 5

Exocytosis and endocytosis are not tightly coupled in BoNT/A poisoned neurons. (a) BoNT/A was labeled with Alexa 488 (green fluorescence). Three week-old cultured mouse spinal cord neurons were incubated with a culture medium containing 1 nM of Alexa 488-BoNT/A for 8 hours at 37°C. Excess Alexa 488-BoNT/A was removed by washings 3 times with fresh culture medium. The cells were incubated with fresh medium for 1 hour and then incubated in medium containing 100 nM Cy3 (red fluorescence) labeled DDV for 16 hours (b). (c) Overlay of red and green showing both BoNT/A (green) and DDV (red) were taken up in the same cell pool, but localized in separate population of endosomes. Fluorescent images were analyzed by Bio-Rad 2000 laser confocol microscope. Alexa 488 was excited at 488 nm line of an argon laser and detected with a 530-nm cutoff filter; Cy3 was excited at 543 nm line of an argon laser and detected with a 565-nm cutoff filter.