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First published online 19 February 2003
doi: 10.1242/jcs.00363

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Observation of keratin particles showing fast bidirectional movement colocalized with microtubules

¹ Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
² Medical Centre for Molecular Biology, Medical Faculty, University of Ljubljana, SI-1000, Slovenia
³ School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK

View larger version (143K): [in a new window]   Fig. 4. Nocodazole inhibits keratin particle movement (Movies 3 and 4). Panel A traces the course of one particle among the several visible keratin F particles in Movie 3, before treatment with nocodazole. Panels B-D are consecutive (20 second interval) frames, depicting the particle's movement (arrow). Panels E and F are two frames, taken several minutes apart, of Movie 4, after the addition of nocodazole (10 µg/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. Images in A-F were obtained by projection of sections 4 and 5.

View larger version (158K): [in a new window]   Fig. 9. Immunofluorescence of the cytoskeleton in untransfected PtK2 cells. Untransfected PtK2 epithelial cells were fixed and stained for actin, tubulin and intermediate filaments as described in Materials and Methods. Representative images are shown of the keratin filament network (K8/K18) (A,B), actin (C) and microtubules (D). Image (A) is acquired with 2x2 binning, whereas (B) is a high-resolution image (1x1 binning) showing keratin filaments in more detail. At higher power, the keratin filaments (only) have a granular appearance, which was not detected with other fixations regimes.

View larger version (164K): [in a new window]   Fig. 1. De novo keratin filament polymerization. To test the capability of the EGFP-K5 fusion protein to form a functional keratin filament network, human lens cells (which otherwise do not express keratins) were transfected with either EGFP-K5/K14 pcDNA3 (A, B) or K5/K14 pcDNA3 (C,D) constructs (see Materials and Methods). At day 1 after transfection a perinuclear ring of keratin filaments is formed in both cases (A, C). At day 2, a keratin filament network is formed in lens cells transfected with the K5/K14 pcDNA3 constructs (D). Extended network formation with the EGFP-K5/K14 pcDNA3 constructs was slightly slower, but by day 3 (B), the network produced was indistinguishable from that of the K5/K14 pcDNA3-transfected cells.

View larger version (83K): [in a new window]   Fig. 2. Dynamic movement of a keratin F particle at the cell periphery (Movie 1). Keratinocytes (NEB-1 cells) transfected with the EGFP-K5 construct were filmed three days after transfection. (A) The course of a fast, keratin F particle. The total distance covered by the particle in 10 minutes is 11 µm. (B-G) Reconstruction of the zig-zag movement of the keratin particle (white arrow). The keratin network may be involved in this non-random movement, as a transient stop by the keratin particle during the saltatory movement (see Movie 1). Because of the variable lengths of the pauses, these images are not consecutive frames of the corresponding time-lapse movie. The longest pause in this sequence (C) lasted two and a half minutes (10 frames). The movie sequence contains 41 time-points with a 15 second interval, and 7 Z sections (200 nm spacing) were taken at each time-point; images were obtained from the projection of sections 5 and 6. Bar, 1 µm.

View larger version (84K): [in a new window]   Fig. 3. Movement of a keratin F particle through the cell (Movie 2). (A) Course of a keratin F particle in a keratinocyte (NEB-1) expressing EGFP-K5, tracked over 10 minutes. (B-H) Individual time-points from this sequence; the position of the particle is indicated by the white arrow. During imaging, the particle travelled a distance of 18 µm. The courses of several other F keratin particles are seen to converge in Movie 2. Some slow, S keratin particles appearing tethered to the keratin network are visible (bottom part of B-H). The S particles moved only slightly, with an oscillatory motion around a central axis. As in Fig. 1, the keratin filament network appears to be involved in the particle movement (B-D); the longest pause (here in D, 3 minutes) occurs when the particle is located over a keratin filament. The movie covers 41 time-points with a 15 second interval, and 9 Z sections (500 nm spacing) were taken for each time-point; images were obtained by projection of sections 5 and 6. Bar, 1 µm.

View larger version (68K): [in a new window]   Fig. 5. Colocalization of keratin particles and microtubules (Movie 5). PtK2 cells were transfected with EGFP-K5 and EYFP–tubulin. Cells were grown to confluence, three days after transfection. Panels A-H show the path of two fast-moving F particles (white and blue arrows). In total, 34 images were acquired with a 5 second time-lapse interval (see Movie 5). 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 are shown in A-H. Two keratin particles (in green, indicated by the white and blue arrows) follow one another along the same path of a microtubule (red, positioned slightly out of the focal plane; see Movie 5).

View larger version (154K): [in a new window]   Fig. 6. Effect of heat shock on PtK2 cells transfected with wild-type (EGPF-K5) and mutant (EGFP-K5 N176S) keratin constructs. (A,B) PtK2 cell expressing EGFP-K5 before (A) and 1 hour after (B) heat shock (15 minutes at 45°C). (C,D) PtK2 cell expressing the EGFP-K5 N176S construct, again before (C) and 1 hour after (D) heat shock. The mutant keratin incorporates well into the endogenous network (C, before heat shock). After heat shock, tiny keratin particles appear in the cell expressing the mutant keratin (D), but not in the wild-type cell (B). 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 (see Movie 6).

View larger version (120K): [in a new window]   Fig. 7. A keratin particle moving away from the centrosome (Movie 7). PtK2 cells were transfected with EGFP-K5 (green) and EYFP–tubulin (red). (A) The microtubule radial array and the centrosome, in close proximity to the nucleus of a transfected cell. An F particle (arrow; B,C) positioned near to the centrosome is seen attaching to a microtubule (D) and sliding along it (E). In total, 21 images were taken with a 30 second time lapse (see Movie 7). Six Z sections were taken per time-point, with a 200 nm spacing. Panel images correspond to section 3. Images were analysed with the edge enhancement tool of softWorX 2.50. Bar, 1 µm.

View larger version (123K): [in a new window]   Fig. 8. A keratin particle moving towards the centrosome (Movie 8). PtK2 cells were transfected with EGFP-K5 N176S (green) and EYFP–tubulin (red). (A) The microtubule radial array and the centrosome, next to the nucleus of a transfected cell. Before imaging, cells were exposed to a heat shock to induce increased keratin particle formation. (B-E) A close-up view of the centrosome region 1 hour after the heat shock. A keratin particle (arrow) is clearly seen moving towards the centrosome and following microtubule tracks. Images from 21 time-points (see Movie 8) were collected (4 Z sections per time-point, 200 nm spacing). Panel images correspond to section 3, processed for edge enhancement. Bar, 1 µm.

View larger version (154K): [in a new window]   Fig. 10. Field emission scanning and transmission EM of untransfected PtK2 cells. Untransfected PtK2 cells were fixed and immunogold labelled (with 8 nm gold particles) for K8, a natural component of the endogenous keratin filament network, as described in Materials and Methods. (A) Scanning EM image to show the degree of cytoskeleton preservation; image acquired using the SE detector (secondary electrons), at 9000x magnification and 10 mm working distance. Bar, 1 µm. (B,C,D) The keratin filament cytoskeleton at high magnification. All images were taken at 130,000x magnification and a working distance of 10.1 mm. Image in (B) was acquired using the SE detector to show maximum detail of the keratin filament surface structure. Image in (C) was acquired with the YAGBSE detector for backscattered electrons, to distinguish gold particles of the immunogold labelling; gold particles appear as white dots on the filaments from the high-energy backscattered electrons, but the image of the filament surface is poor. Both SE and YAGBSE images are combined in (D), where a keratin particle attached to the side of a keratin filament can be clearly seen (white arrow). Bar, 100 nm. (E) Transmission EM image of cell after the same fixation as in (A-D), also labelled with anti-K8 monoclonal antibody CAM 5.2. Immunogold labelling was performed before resin embedding and labelling is seen (black dots) along the keratin filaments. Keratin particles are seen adhering to the filaments (black arrows). Note that microtubules (*) are unlabelled. Bar, 100 nm.