TTI motility is dependent on an intact microtubule cytoskeleton. (A and B) HeLa p24-YFP cells were incubated at 15°C for 30 min and then at 37°C for 5 min in the presence of 10 μM nocodazole before fixing and immunostaining for either α-tubulin (A) or GM130 and TGN46 (B). Under these conditions the microtubule cytoskeleton was almost totally dispersed; however, the Golgi complex and TGN retained their juxta-nuclear localization. Remnants of microtubules often colocalized with p24-YFP vesicular and tubular structures (inset in A, arrowheads). Asterisks indicate p24-YFP expressing cells. (C) HeLa cells stably expressing p24-YFP were incubated at 15°C for 30 min in the presence of 10 μM nocodazole and then incubated at 37°C for 5 min. Short nonmotile TTIs can be observed (arrowheads). (D) HeLa cells stably expressing p24-YFP were incubated at 15°C for 30 min in the presence of 10 μM nocodazole, and then the nocodazole washed out and the cells were incubated at 37°C for 5 min. Longer TTIs can now be observed (arrowheads). (E) HeLa cells stably expressing p24-YFP were incubated as described above, and the frequency of cells in the population with distinct TTIs was determined. Control cells were incubated at 15°C for 30 min, and then at 37°C for 5 min, all in the absence of nocodazole. (F–I) Analysis of vesicular versus TTI carriers in individual cells incubated under the nocodazole treatments described above. Randomly selected distinct peripheral structures were counted and determined to be either vesicular or tubular. (F) The percentage of tubular structures under the various conditions is shown. (G) The percentage of those structures that were motile is shown. Gray bars are vesicular carriers; black bars are tubular carriers. (H) Only motile structures were counted, and these were determined to be either vesicular or tubular, allowing their relative contribution to motility/transport to be determined. Gray bars are vesicular carriers; black bars are tubular carriers. (I) The distance moved by the motile structures was determined. Gray bars are vesicular carriers; black bars are tubular carriers. Error bars in E indicate mean and SD between three replicate experiments. Error bars in F–I indicate mean and SD between individual cells. Bars, 10 μm. Movies accompany C and D.