The Journal of Neuroscience, November 23, 2005, ():
Phorbol Esters Target the Activity-Dependent Recycling Pool and Spare Spontaneous Vesicle Recycling
J. Neurosci. Virmani et al.
25: 10922
Supplemental data
Files in this Data Supplement:
- supplemental material -
Supplementary Figure. Raw traces from a representative experiment analyzed in Figure 3. In these experiments, we loaded synapses with FM2-10 dye using 47 mM K+ / 2 mM Ca2+ application for 90s. After a 10-minute washout period, we imaged synapses during a 15 s application of 500 mM sucrose solution to measure the size of the RRP. We then continued to image during maximal destaining achieved by application of multiple rounds of 90 mM K+ solution. This complete destaining provided us with the total recycling vesicle pool size in these synapses (Top panel). After a brief 5-minute wash, the synapses were treated with either 1μM PMA or 4α-PMA for 2 minutes and then washed again for 5 minutes to remove any residual PMA or 4α-PMA. We then performed a second round of 47 mM K+ loading and destaining, similar to that described above to determine what affect PMA has on this vesicle pool. Under these conditions the treatment of synapses with PMA increased sucrose dependent dye release (Bottom panel) as well as increased the ratio of the dye released by 15s of sucrose to the total pool (Δsuc/ΔF) plotted with respect to the total recycling pool (ΔF) (Figure 3E).
