Vps8Dp concentrates at junctions between adjacent vacuoles
A: Live imaging of numerous large Dop1p-labeled vacuoles in cells
expressing Dop1p-mNeon, following hypo -osmotic shock. The cell
shown was imaged to capture a cross-sectional view, in a time-lapse
video with 4 sec frame intervals (Movie 6). Four successive images
extracted from the video are shown. Growing cell cultures were
hypo-osmotically challenged with 10 mM Tris-HCl buffer, pH 7.4 and
immediately immobilized for imaging using CyGEL, as described in
Materials and Methods. Videos were captured using a Zeiss Axio Observer
7 system.
B: Live imaging of the numerous Dop1p-labeled vacuoles followinghyper -osmotic shock in cells expressing Dop1p-mNeon. The cell
shown was imaged to capture a cross-sectional view, in a time-lapse
video with 0.15 sec frame intervals (Movie 7). Four successive images
extracted from the video are shown. Growing cell cultures were
hyper-osmotically challenged with 10 mM sorbitol and immediately
immobilized for imaging using CyGEL, as described in Materials and
Methods. Videos were captured using a Marianas spinning disc confocal
microscope.
C: Live imaging of numerous large Vps8Dp-labeled vacuoles followinghypo -osmotic shock in cells expressing Vps8Dp-mNeon. The cell
shown was imaged to capture a cross-sectional view, in a time-lapse
video with 0.5 sec frame intervals (Movie 8). Four successive images
extracted from the video are shown. Cells were treated as described in
Fig. 5A. Videos were captured using a Marianas spinning disc confocal
microscope.
D-E: Imaging of extracellular Vps8Dp-labeled vacuoles that leaked from
ruptured cells expressing Vps8Dp-mNeon.
D. Vps8Dp localizes densely at contact site between adjacent vacuoles,
which in some cases are sites of subsequent membrane fusion (arrowhead).
Four successive images extracted from Movie 9 are shown.
E: Vps8Dp re-distributes at the time that two vacuoles come into
contact. The two large vacuoles shown were separated by
~1µ at the beginning of the video (t=0) (Movie 10). When
they moved into contact starting at t=10s, Vps8Dp accumulated at the
junction (boxed). A second example of this phenomenon is shown in Movie
11.
F. Plot of fluorescence intensity change with time for the boxed area
shown in Fig. 5E, in Movie 10. The data were plotted using GraphPad
Software Prism. The three red arrows correspond to the images shown in
panel E.
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