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Movie 1
Dynamic movement of a keratin F particle at the cell periphery. Keratinocytes (NEB-1 cells) transfected with the EGFP-K5 construct were filmed three days after transfection. The movie presents the zig-zag course of a fast keratin F particle (see arrow). The total distance covered by the particle in 10 minutes is 11 mm. The keratin network may be involved in this non-random movement, as a transient stop by the keratin particle during the saltatory movement. The particle appears to pause in certain positions. The length of the pauses varies, with the longest lasting two and a half minutes (10 frames). The movie sequence contains 41 time points with a 15 second interval, and seven Z sections (200 nm spacing) were taken at each time point; the individual frames of the movie were obtained by projecting sections 5 and 6 of each time point.
Movie 2
Movement of a keratin F particle through the cell. The course of a keratin F particle (arrow) in a keratinocyte (NEB-1) expressing EGFP-K5 was tracked over 10 minutes. During imaging, the particle travelled a distance of 18 mm. The courses of several other F keratin particles are seen to converge. Some slow S keratin particles that appear to be tethered to the keratin network are visible as well (bottom part of the panel). The S particles moved only slightly, with an oscillatory motion around a central axis. As in Movie 1, the keratin filament network appears to be involved in the particle movement; the longest pause here is 3 minutes and occurs when the particle is located over a keratin filament. The movie covers 41 time points with a 15 second interval, and nine Z sections (500 nm spacing) were taken for each time point; the frames of the movie were obtained by projecting sections 5 and 6 of each time point.
Movie 3
Nocodazole inhibits keratin particle movement. The keratin intermediate filament network of a keratinocyte expressing EGFP-K5 was filmed 3 days after transfection. Movie 3 presents the course of one (arrow) of the several visible keratin F particles prior to treatment with nocodazole. Movie 4 shows the same cell after the addition of nocodazole (10 mg/ml). Nocodazole stops both F keratin particle movement and keratin filament wave-like motion (compare Movies 3 and 4). Both movies were compiled from 16 images taken at 20 second intervals. Z sections were taken with a 200 nm spacing. The individual frames of the movie were obtained by projecting sections 4 and 5 of each time point.
Movie 4
Nocodazole inhibits keratin particle movement. The keratin intermediate filament network of a keratinocyte expressing EGFP-K5 was filmed 3 days after transfection. Movie 3 presents the course of one (arrow) of the several visible keratin F particles prior to treatment with nocodazole. Movie 4 shows the same cell after the addition of nocodazole (10 mg/ml). Nocodazole stops both F keratin particle movement and keratin filament wave-like motion (compare Movies 3 and 4). Both movies were compiled from 16 images taken at 20 second intervals. Z sections were taken with a 200 nm spacing. The individual frames of the movie were obtained by projecting sections 4 and 5 of each time point.
Movie 5
Colocalization of keratin particles and microtubules. PtK2 cells were transfected with EGFP-K5 (green) and EYFP-tubulin (red). Cells were grown to confluence 3 days after transfection. The movie tracks the movement of two fast moving F particles (arrows). In total, 34 images were acquired with a 5 second time lapse interval. Six Z sections with a 100 nm spacing were taken per time point and subsequently analysed using the edge enhancement tool (represents filaments as tubes rather than lines) of softWorX 2.50 software. The resulting images for section 1 were used to compile this movie. The two keratin particles (arrows) follow one another along the same path of a microtubule (red, positioned slightly out of the focal plane).
Movie 6
Effect of heat shock on PtK2 cells transfected with a mutant (EGFP-K5 N176S) keratin construct. PtK2 cells expressing the EGFP-K5 N176S construct were filmed 1 hour after heat shock (15 minutes at 45°C). After heat shock, tiny keratin particles appear only in cells expressing the mutant keratin. Again, two types of particles can be distinguished: fast moving F particles and larger slow S particles that appear to be tethered to the keratin filament network..
Movie 7
A keratin particle moving away from the centrosome. PtK2 cells were transfected with EGFP-K5 (green) and EYFP-tubulin (red). The movie shows the close up view of the centrosome region. An F particle (arrow) positioned near to the centrosome is seen attaching to a microtubule and sliding along it. In total, 21 images were taken with a 30 second time lapse. Six Z sections were taken per time point, with a 200 nm spacing. The individual frames of the movie correspond to section 3 and were analysed with the edge enhancement tool of softWorX 2.50.
Movie 8
A keratin particle moving towards the centrosome. PtK2 cells were transfected with EGFP-K5 N176S (green) and EYFP-tubulin (red). Prior to imaging, cells were exposed to a heat shock to induce increased keratin particle formation. As in movie 7, we show a close up view of the centrosome region, but 1 hour after the heat shock. A keratin particle (arrow) is seen moving towards the centrosome and following microtubule tracks. Images from 21 time points were collected (four Z sections per time point, 200 nm spacing). The frames of the movie correspond to section 3, processed for edge enhancement.
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