Fluorescence imaging of YFP-hVps35 in live cells. YFP-hVps35 is present in punctate and tubular structures that are often associated with dark vacuolar profiles. The tubule marked by the arrowhead extends, detaches from a vacuole, and heads towards the juxtanuclear area. The dark vacuole moves along with the YFP-hVps35-positive structure before they separate. This experiment was performed on a spinning disk confocal microscope (Ultraview LCI confocal scanner; PerkinElmer) equipped with a stage heated to 37°C, and a 63 Å/1.4 NA objective. YFP-hVps35 was excited with a 488-nm line and imaged through a 500 LP emission filter. Frames were recorded every 0.4 s.
