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Video 1 (5.5MB)
Corresponds to Fig. 4a. Cav^DGV vesicles are immobile and nonacidic vesicles (real-time 28 min).
BHK cells were transfected with GFP-tagged cav^DGV. After 24 h, the growth media was replaced with Air medium containing 50 nM of lysoTraker (a marker for acidic compartments). The cells were observed in an Olympus BX60 microscope with a 60x water immersion lens, and the images were captured using IPLab software with a CCD camera (Hamamatsu). Then, the automatic capture was set at 20-s intervals. After the automatic capture in the first channel, another image was immediately captured in the second channel with a delay of 2 s. A total of 84 frames were captured during 28 min. All images were equally processed using Adobe Photoshop 5.0 to enhance contrast and adjust the apparent intensity in the dual color movie. The lysoTracker-labeled vesicles showed rapid bidirectional motility (up to 5 mm/min). In contrast, the cav^DGV-GFP-labeled vesicles showed no significant motility in any axis.