Figure 1D1E

1
Figure 1D/1E
Tucker W., Weber T., Chapman E., Reconstitution
of Ca2-Regulated Membrane Fusion by
Synaptotagmin and SNAREs

• Chintan Hossain

Nov. 2, 2005
2
Setup

• Two vesicles types
• t-SNARE vesicles w/ syntaxin SNAP-25
• v-SNARE vesicles w/ syb
• Vesicles fused if syt Ca2 present
• Figure 1D 1E - vary of syntaxin/SNAP-25
  complexes

3
Figure 1D

• Prepare v-SNARE vesicles with different of
  syntaxin SNAP-25 proteins
• Run SDS-PAGE on the vesicles
• Visualize with a Coomassie stain

4
Procedure for 1E

• Combine v-SNARE and t-SNARE vesicles
• Add Ca2 and syt
• Measure FRET fluorescence as a function of syt
• Compare plots of fluoresence vs. syn for
  different syntaxin SNAP-25 densities

5
Figure 1E

• Amount of vesicle fusion saturates
• syt required for saturation increases as more
  syntaxin SNAP-25 are added

6
Conclusion

• Concentration of syt needed to saturate vesicle
  fusion increases as more syntaxin and SNAP-25 are
  added.
• Suggests that syt acts by direct interaction with
  syntaxin and SNAP-25.

7
The End