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doi: 10.1242/10.1242/jcs.00096

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Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases

Department of Anatomy, Johannes Gutenberg-University, Becherweg 13, 55128 Mainz, Germany

View larger version (105K): [in a new window]   Fig. 1. Double fluorescence microscopy of methanol/acetone-fixed AK13-1 cells to detect the distribution of the CK13-EGFP chimera HK13-EGFP (A-D) together with human CK5 using polyclonal rabbit antibodies (A'-D') before (A,A') during (B,B',C,C') and 50 minutes after a 10 minute treatment with 5 mM OV (D,D'). Note the progressive loss of CKFs during OV incubation accompanied by formation of granules throughout the cytoplasm, which remain in close association with residual filaments. Both processes are reversed upon removal of the drug, yielding a fine CKF cytoskeleton. Bars, 10 µm.

View larger version (177K): [in a new window]   Fig. 2. Microscopic analyses of OV-treated AK13-1 cells. (A-D) Inverse fluorescence micrographs of AK13-1 cells that were treated with 5 mM OV for 2 minutes (A,B), 10 mM OV for 6 minutes (C) or not at all (D) demonstrate details of the distribution of the CK13-EGFP chimera HK13-EGFP. The overview in (A) shows multiple granular thickenings along filamentous structures and granules that are in close apposition to residual filaments. B and C present further details of granules, which are either part of filaments, in the near vicinity of filaments, or without apparent connection. C depicts, in addition, variously shaped CK formations ranging from spheroidal bodies to elongated rod-like structures. Note also the perseverance of desmosome-anchored filaments (arrowheads in B), whereas granulation has proceeded considerably in other nearby CKFs. Bars, 2 µm. (E-H) Electron microscopy (E,F) and immunoelectron microscopy (G,H) of AK 13-1 cells depicting different stages of CKF network breakdown during OV treatment (2 mM for 3 minutes in E,G,H; 10 mM for 2 minutes in F). Note the abundant dense filament bundles with local densities (+) and the multiple amorphous granules (*) that are often in association with residual filaments (E, inset in E,G) but become separated during later stages (F). Normal-appearing microtubules are present while filament aggregation is already in progress (arrowheads in E). For immunoelectron microscopy, antibodies against EGFP were used in combination with 1 nm gold-labeled secondary antibodies and the silver amplification technique. The label is primarily detectable on the surface of dense filament bundles and aggregates, whereas comparatively little labeling is seen within these structures. Bars, 200 nm.

View larger version (164K): [in a new window]   Fig. 3. Fluorescence microscopy of methanol/acetone-fixed AK 13-1 cells detecting the chimera HK13-EGFP (A-H) together with microtubules using anti -tubulin antibodies in combination with Texas-Red-labelled secondary antibodies (A'-D') or actin filaments employing Texas-Red-coupled phalloidin (E'-H') prior to OV incubation (A,A',E,E'), during OV treatment (2 minutes in B,B',F,F' and 10 minutes in C,C',G,G'), and after a 50 minutes recovery period following a 10 minute OV-incubation (D,D',H,H'). OV concentration was 5 mM. Note the partial loss of microtubules and the increased peripheral localisation of actin during drug treatment, both of which are reversed after drug removal and do not reflect the dramatic changes of the CKF system. Bars, 10 µm.

View larger version (157K): [in a new window]   Fig. 4. Fluorescence microscopy depicting the distribution of HK13-EGFP (A-K) together with plectin (A'-E'; monoclonal antibodies 10F6) or 14-3-3 (F'-K') in methanol/acetone-fixed AK13-1 cells in the absence of OV (A,A',F,F'), after a 2 minute OV treatment (5 mM; B,B',G,G'), a 10 minute OV treatment (5 mM; C,C',H,H'), after 2 or 3 hours of okadaic acid incubation (0.1 µg/ml; D,D',I,I') or during mitosis (E,E',K,K'). Note the increased co-distribution of plectin and CKs during OV treatment and mitosis and the lack of plectin staining of okadaic-induced granules, which is in contrast to 14-3-3 distribution exhibiting the reverse behavior. Bars, 10 µm.

View larger version (10K): [in a new window]   Fig. 5. Whisker-box plots showing colocalisation between HK13-EGFP fluorescence and plectin immunostaining in AK13-1 cells using Pearson's correlation coefficient. For each group, eight typical double-labelled cells were analysed. The top and bottom of each box delineate the upper and lower 25 percentiles, respectively, the center line demarcates the median and the dots depict the minimum and maximum values. UC, untreated interphase cells; OV, cells treated with 5 mM OV for 10 minutes; OVR, cells treated with 5 mM OV for 10 minutes followed by incubation in OV-free medium for additional 50 minutes; MIT, mitotic cells (late prometaphase); OA, cells treated with 0.1 µg/ml okadaic acid for 3 hours.

View larger version (137K): [in a new window]   Fig. 6. Fluorescence microscopy of cells stably expressing fluorescent CK chimeras depicting CKF network disruption upon treatment with OV. (A) Inverse contrast fluorescence micrographs taken from living AK13-1 cells at 0.2 minute intervals during OV treatment (10 mM) showing HK13-EGFP fluorescence. The image series starts 1 minute after addition of OV and displays the rapid disruption of the CKF network. Note that the breakdown of CKFs into small granules (arrows) is paralleled by dissociation of desmosome-anchored filaments (arrowheads). (B-D) Fluorescence microscopy of hepatocellular carcinoma-derived PLC cells PK18-5 stably expressing fluorescent fusion protein HK18-YFP. Cells were fixed with methanol/acetone prior to (B) or 15 minutes after addition of 50 mM OV (C). Note the loss of filaments and the formation of rods and granules in C. D shows inverse contrast images at high magnification that were taken from a live cell recording (Movie 1; recording intervals 1 second) demonstrating details of OV-induced CKF-disruption. Note the straightening of CKFs (arrows) prior to fragmentation into rods and granules. Bars, 10 µm in (A-C) and 1 µm in (D).

View larger version (167K): [in a new window]   Fig. 7. Time-lapse fluorescence microscopy of AK13-1 cells depicting the altering distribution of HK13-EGFP fluorescence after OV addition (10 mM). The inverse contrast images are taken from Movie 2 (recording intervals 8 seconds; available at jcs.biologists.org/supplemental). Note the formation of fine granules within filaments reaching a maximum 12 minutes after OV addition and the gradual re-integration of these granules into CKFs with almost no thickenings remaining by 38 minutes (arrows). Separating desmosome-anchored filaments are demarcated by arrowheads. Bar, 10 µm.

View larger version (189K): [in a new window]   Fig. 8. Fluorescence micrographs taken from a time-lapse recording of AK13-1 cells monitoring the distribution of fusion protein HK13-EGFP following a transient 3 minute treatment with 10 mM OV. Note the reformation of an extended CKF network within less than 30 minutes with no remaining granules and a homogenous composition. The entire sequence is provided as Movie 3 (recording intervals 15 seconds; available at jcs.biologists.org/supplemental). Elongation and fusion of granules and their integration into a fine network is shown at high magnification in the lower panel (corresponding to the boxed area in upper panel). The dynamics of this process can be best appreciated in the corresponding Movie 4. Bar, 10 µm in upper panels and 1 µm in lower panels.

View larger version (56K): [in a new window]   Fig. 9. Gel electrophoretic analysis of CKs in AK 13-1 cells treated with OV. (A) A picture of an immunoblot of SDS polyacrylamide gel (10%) containing high salt pellet fractions (P) and equal amounts of corresponding supernatant fractions (S) that were obtained either from untreated control cells (C), cells treated with either 5 mM OV for 10 minutes (OV) or 0.1 µg/ml okadaic acid for 105 minutes (OA) and enriched mitotic cells (mit) after reaction with CK antibody Ks 13.1 and secondary HRP-coupled antibodies in combination with an enhanced chemiluminescence system. Note the presence of CK13 (lower band) and HK13-EGFP (upper band) in the soluble fraction of cells treated with okadaic acid and in mitotic cells in contrast to untreated and OV-treated cells. The position and relative molecular mass of co-electrophoresed marker proteins are given in units of 1000 in left margin. (B,C) Detection of Coomassie brilliant blue stained polypeptides contained in high salt pellet fractions after separation by two-dimensional gel electrophoresis obtained from untreated control cells (B) and cells treated with 5 mM OV for 10 minutes (C). Note the similar distribution of CK polypeptides (*, positions of chimera HK13-EGFP) with some minor differences. (D) Immunoblot reaction of high salt pellet fractions of AK13-1 cells without any treatment or after incubation in 5 mM OV for 10 minutes (OV) and 0.1 µg/ml OA for 4 hours (OA) using phospho-epitope specific antibodies LJ4 detecting CK8 pS73, 5B3 reacting with CK8 pS431, and IB4 detecting CK18 pS33 as well as CK antibody L2A1 detecting all CK8, 18 and 19 polypeptides irrespective of their phosphorylation status. The position and relative molecular mass of marker proteins are shown on the left.

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