Movie 3

Counting of punctate structures and object-based co-localization analysis. Acquired confocal images of the bottom half of embryos (21 z-axis planes with 2 μm increments) for 488 nm (EGFP-hRAB5A) and 561 nm (mGlyT1a-mCherry) were processed with background subtraction and a Gaussian blur filter using Fiji software. Then, thresholding, three dimensional object reconstruction, and counting of the objects were processed on JACoP plugin of Fiji. The number of colocalized centers of mass (GlyT1a)-particle (RAB) coincidences was counted as the number of colocalized particles.