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Files in this Data Supplement:
Fig. S1. Monitoring the effects of MT depolymerisation on the ER network in Xenopus A6 and VERO cells. Upper panels: first frames of a movie taken of Xenopus A6 cells expressing GFP-ER and mRFP-actin at 25°C, 2-4 minutes after the addition of nocodazole to the medium. Lower panels: first frames of movies taken of VERO cells expressing GFP-ER along with RFP (left panel) or RFP-KHCct (right panel) and imaged at 37°C, 2-4 minutes after the addition of nocodazole to the medium. Scale bar: 20 µm. The boxed regions are shown in supplementary material Movie 6.
Movie 1. Effect of MT and actin depolymerisation on ER movement. VERO cells expressing GFP-ER were treated with nocodazole or cytochalasin D, or left untreated (control). Cells were imaged for 30 seconds at 10 frames/second, and stills are shown in Fig. 2. Movies were made in ImageJ from frames 50-149 with Sorensen3 normal compression. Playback is set at 30 frames/second (3× real time). Scale bar: 10 µm.
Movie 2. TAC-dependent ER motility. VERO cells expressing GFP-ER and Tomato-EB3 were imaged for 2.5 minutes at 2 frames/second, and stills are shown in Fig. 3A. The movie was made in ImageJ from frames 203-302 with MPEG-4 high compression. Playback is set at 30 frames/second (15× real time). Scale bar: 5 µm.
Movie 3. Inward and outward motility of the ER tubules. VERO cells expressing GFP-ER were imaged for 30 seconds at 10 frames/second, and 100 frames are shown. Frames I and II correspond with the boxes in Fig. 3B. Movies were compressed in ImageJ using the Sorensen3 high setting, and playback is 30 frames/second (3× real time). Scale bar: 10 µm.
Movie 4. Minus-end-directed movement of an ER tubule. VERO cells expressing GFP-ER and dtTomato-EB3 were imaged at 2 frames per second, and 36 individual and colour-merged frames are shown, played back at 6 frames/second (3× real time). Selected individual frames are presented in Fig. 3E. The movie was saved in QuickTime without compression. Scale bar: 10 µm.
Movie 5. Effect of the kinesin-1 fragments on ER motility. VERO cells expressing GFP-ER and either RFP, RFP-DTC, RFP-BTC, RFP-KHCct or RFP-KHCtail were imaged for 30 seconds at 10 frames/second. The first and last frames from the full-length movie are shown in Fig. 5. Frames 46-149 were compressed in ImageJ with Sorensen3 compression set at normal, and playback is 30 frames/second (3× real time). Scale bar: 10 µm.
Movie 6. Effects of MT depolymerisation on the ER network in Xenopus A6 and VERO cells. Upper panels: Xenopus A6 cells expressing GFP-ER and mRFP-actin were imaged at 25°C 2-4 minutes after the addition of nocodazole to the medium. The movie was collected at one frame per 6 seconds, but every second frame is shown at a playback speed of 10 frames/second (120× real time) with Sorensen3 compression set at normal. Lower panels: VERO cells expressing GFP-ER along with RFP (left panel) or RFP-KHCct (right panel) and imaged at 37°C, as described above. Scale bar: 10 µm. Full-frame images of these cells are shown in Supplementary material Fig. S1.
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