Fig. 9. Dynein is required for vesicle formation and transport of Spitz-GFP in S2 cells. (A) Spitz-GFP distribution in S2 cells is largely restricted to the ER (see also, Tsruya et al., 2002). A single 1 µm section is shown to highlight the membrane network. (B) Coexpression of Spitz-GFP and Star-HA shifts the distribution of Spitz-GFP into vesicles that exhibit transient movements through the cytoplasm. A projection of sequential images shows the tracks of vesicle movements. Red bars highlight the position of several of these tracks. See also supplementary material Movie 1. (C) RNAi depletion of Dhc reduces vesicle number and inhibits motility. No tracks of moving vesicles are seen in this projection of sequential images. See also the graphs in E and F. (D) Dynein is present throughout the cytoplasmic compartment, and colocalizes with a subpopulation of Spitz-GFP vesicles after coexpression of both Spitz-GFP and Star-HA. An image stack of four optical sections shows both Dhc (red) and Spitz-GFP (green) channels. Arrows highlight the positions of some of the overlapping signals, which appear yellow. The inset is enlarged by a factor of two. Scale bar: 10 µm (applies to all images). (E) RNAi depletion of Dhc decreases the number of Spitz-GFP vesicles per cell. Graph shows the average number of Spitz-GFP vesicles observed in a single focal plane. Error bars depict ± s.e.m. Wild type, 339 vesicles from 9 cells; Dhc RNAi, 356 vesicles from 25 cells (P<0.001). (F) Depletion of Dhc also decreases the frequency of transport events of Spitz-GFP vesicles. The graph shows the average percentage of vesicles in each cell that are motile, calculated from the same cells used in E. Error bars depict ± s.e.m.