Two kymographs were generated for each time-lapse; one for the synapse and one for the cell body.
YFP expression was verified by fluorescence imaging and it is shown in the first panel for each time-lapse series.
The synapse and the cell body layers were then separately selected, resliced (Image>Stack>Reslice), and Z-projected (Sum slices) to generate two separate kymographs for each time-lapse.
For each time-lapse movie, mitochondria or autophagic vacuoles were manually tracked and transport parameters were generated using the ImageJ software plug-in Multiple Kymograph, submitted by J. Rietdorf and A. Seitz (European Molecular Biology Laboratory, Heidelberg, Germany).
For each well, time lapse movies were obtained at four fields of view.
Except for the longest periods, each time-lapse fluorescence series extended for at least two full oscillation periods.
See Video 5 for full time lapse sequence.
For time lapse imaging ~1 cm2 of agar was cut around each 3 DAG plant and the TUB6 GFP-expressing seedlings were aligned on a peTUB6 GFP-expressingered seedlings mounted in werer.
These long exposures bleached the GFP signal, precluding the repeated exposures necessary for time lapse movies, which we used to quantitate the fluorescence for the exocyst proteins.
For time lapse imaging, live cells were housed in a heated incubation chamber with constant CO2.
