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[HELP with Viewing Videos][To Article]
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Videos |
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| Fig.
1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 |
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All movies are series of fluorescent images. Channels in color movies are represented as red and green, with overlap in yellow. Perception of red, green, and yellow depends on monitor settings (See the authors' viewing instructions). "Grey" movies show the individual channels side-by-side.
All movies are in QuickTime format. For optimal playback, edit your browser preferences to launch the QuickTime MoviePlayer as an application, rather than viewing within the browser using the plugin.
Movies are compressed; to achieve smooth playback they must uncompress into
RAM. Set them to repeat (Movie > Loop menu selection) and allow
them to play through more than once. For best results on MacOS platforms, allocate
more memory to the QuickTime movie player application (File > Info > Memory).
For machines with less memory, play a selection only to improve performance:
make a selection by holding the shift key down while moving the slider through
the frames of interest, then select Movie > Play Selection Only.
For additional information on optimizing movie playback, please refer to the
authors' viewing instructions.
The movies have been adjusted for best display on a Macintosh monitor, gamma 1.8, white point correction 6500 or 9300K. You may need to adjust the brightness, contrast, and gamma correction for the best viewing. Fainter structures may only be visible after correcting for your monitor. Viewing is best in a darkened room.
All movies are played at 15 fps, which corresponds to 15 times real speed.
For optimal viewing of fusion events, movies should be played both forwards and backwards.
 Figure 1. Live
cell visualization of the late exocytic pathway by combined EPI and TIR.
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| b | Fig_1b.mov |
Two cells imaged by epi-illuminated wide-field (EPI) and
total internal reflection (TIR) or evanescent wave microscopy. Organelles and TCs away from the plasma membrane (>100 nm) are visible by EPI (red) only. Once TCs approach the plasma membrane, they also become visible by TIR (green). Note that initially red TCs turn yellow and then green when about to dock and fuse with the plasma membrane. A transport container (TC), moving away from the Golgi and then undergoing partial fusion with the plasma membrane, is marked by an arrow and shown enlarged in Fig_1c.mov. Two successive fusion events in close proximity are circled and enlarged in Fig_1d.mov. The whole sequence is 300 frames (300 s). Frame width, 85 µm. |
| c | Fig_1c.mov |
A TC exits from the Golgi and moves to the plasma membrane,
where it partially fuses. Selected region of Fig_1b.mov enlarged twofold. Frame width, 18.0 µm. |
| d | Fig_1d.mov |
Two TCs undergo successive fusion in close proximity.
Selected region of Fig_1b.mov enlarged twofold. Two TCs undergo successive fusion in close proximity. Only the second undergoes complete fusion. Frame width, 8.9 µm. |
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Fig_2_grey.mov |
TCs either fuse, move across, or hover above the plasma
membrane without undergoing fusion. TCs were observed to either fuse, move across, or hover (tethered, but with motion in the z-axis) above the plasma membrane without undergoing fusion (left panels). A differential analysis (right panels) allows to distinguish these possibilities. A true fusion event results in sequential white and black dots that expand as donutlike rings. TCs moving across the field result in black and white dots that keep their respective orientation. A TC hovering above the plasma membrane yields an assymetric pattern in which the relative orientation of the dots changes over time. The whole sequence is 80 frames (10 s) and the movie plays at 8 fps. Frame width, 2.12 µm. |
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| Figures 3 and 4.
Fusion of TCs with the plasma membrane monitored by EPI. |
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Fig_3and4_color.mov |
Small and large fusions of TCs with the plasma membrane
monitored by EPI. TCs fusing at the plasma membrane are heterogenous in size and kinetics.While there is substantial activity in the central region, there is hardly any fusion event occurring in the cell periphery. Events further analyzed in Figures 3 and 4 are highlighted (>). The whole sequence is 700 frames (700 s). Frame width, 70.2 µm. |
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| Figure 5. Large tubularvesicular TCs can
undergo partial fusion at the tip. |
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| a | Fig_5a.mov |
A single cell imaged by EPI and TIR. Fluorescence observed within the cell by EPI is in red and fluorescence observed at the plasma membrane by TIR is in green. A tubular-vesicular TC undergoing partial fusion at the tip of a tubular extension is highlighted (> above the central Golgi region) and enlarged in Fig_5b.mov. Another tubular-vesicular TC ( > below the central Golgi region) that approaches the plasma membrane, but retracts without fusing is enlarged in Fig_5c.mov. The whole sequence is 300 frames (300 s). Frame width, 86.3 µm. |
| a | Fig_5b.mov |
A tubularvesicular TC undergoing partial fusion at the
tip of a tubular extension Selected region of Fig_5a.mov enlarged twofold. A TC approaches the plasma membrane by a tubular extension, tethers with the tip of the tubule, and fuses (>) at that site. Fusion is transient and incomplete. The tubular extension detaches and collapses back into a globular structure. Frame width, 11.5 µm. |
| c | Fig_5c.mov |
A tubular-vesicular TC approaches the plasma membrane,
but retracts without fusing Selected region of Fig_5a.mov enlarged twofold. Frame width, 10.2 µm. |
Correspondence to: simons{at}embl-heidelberg.de
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