Research Article 4133 Induction of rapid and reversible cytokeratin filament network remodeling by inhibition of tyrosine phosphatases

Pavel Strnad, Reinhard Windoffer and Rudolf E. Leube* Department of Anatomy, Johannes Gutenberg-University, Becherweg 13, 55128 Mainz, Germany *Author for correspondence (e-mail: [email protected])

Accepted 7 August 2002 Journal of Cell Science 115, 4133-4148 © 2002 The Company of Biologists Ltd doi:10.1242/jcs.00096

Summary The cytokeratin filament network is intrinsically dynamic, throughout the cytoplasm without apparent correlation to continuously exchanging subunits over its entire surface, alterations in the actin- and tubulin-based systems. while conferring structural stability on epithelial cells. Interestingly, the short-lived and rather small However, it is not known how cytokeratin filaments are orthovanadate-induced cytokeratin granules contained the remodeled in situations where the network is temporarily cytoskeletal crosslinker plectin but lacked the cytokeratin- and spatially restricted. Using the tyrosine phosphatase solubilising 14-3-3 proteins. By contrast, the long-lived and inhibitor orthovanadate we observed rapid and reversible larger cytokeratin aggregates generated after treatment restructuring in living cells, which may provide the basis with the serine/threonine phosphatase inhibitor okadaic for such dynamics. By examining cells stably expressing acid were negative for plectin but positive for 14-3-3 fluorescent cytokeratin chimeras, we found that proteins. Taken together, our observations in living cytokeratin filaments were broken down and then formed orthovanadate-treated interphase cells revealed modes of into granular aggregates within a few minutes of cytokeratin remodeling that qualify as basic mechanisms orthovanadate addition. After drug removal, gradual re- capable of rapidly adapting the cytokeratin filament incorporation of granules into the filament network was cytoskeleton to specific requirements. observed for aggregates that were either part of residual filaments or stayed in close apposition to remaining Movies available on-line filaments. Even when cytokeratin filaments were no longer detectable, granules with low mobility were still able to re- Key words: Tyrosine phosphorylation, Keratin, Intermediate establish a cytokeratin filament network. This process took filament, Plectin, 14-3-3, Live cell imaging, Green fluorescent less than 30 minutes and occurred at multiple foci protein

Introduction Coulombe and Omary, 2002) and to drug resistance (Bauman Intermediate filaments (IFs) of the cytokeratin (CK) type are et al., 1994). major stabilising elements of the epithelial cytoskeleton (Fuchs In terms of molecular diversity, the IF polypeptides of the and Cleveland, 1998; Coulombe and Omary, 2002). The CK type are certainly the most complex, comprising more than numerous genodermatoses caused by CK gene mutations are 50 members, which are expressed as pairs of type I and type proof of this important property of the CK filament (CKF) II isoforms in a differentiation-dependent manner (Moll, 1998; system (Irvine and McLean, 1999). This view is further Hesse et al., 2001; Coulombe and Omary, 2002). In contrast to supported by analyses of knockout mice, which present the other major cytoskeletal filaments, IFs form spontaneously variable degrees of epithelial deficiencies and increased and rapidly in vitro without energy consumption, auxiliary sensitivity to various forms of stress (Galou et al., 1997; Magin, proteins or other factors (Hofmann, 1998; Herrmann et al., 1998). It is evident, however, that CK functions extend beyond 1999; Herrmann and Aebi, 2000; Coulombe and Omary, 2002). the mere structural maintenance of epithelial integrity. Thus, In the case of CKs, parallel heterodimers assemble in an CKs influence the availability of regulatory molecules such as antiparallel fashion into tetramers, which associate apoptosis-inducing factors, heat shock proteins or signalling longitudinally and laterally into protofilaments to form the molecules (Omary et al., 1998; Perng et al., 1999; Inada et al., bona fide 8-12 nm IFs (Herrmann et al., 1999; Herrmann and 2001; Toivola et al., 2001). Therefore, CKs affect the Aebi, 2000; Parry and Steinert, 1999; Coulombe and Omary, sensitivity of cells to proliferative and apoptotic stimuli 2002). In vivo, soluble tetrameric and larger oligomeric (Paramio et al., 1999; Paramio et al., 2001; Gilbert et al., 2001; precursors have been identified which presumably incorporate Inada et al., 2001; Toivola et al., 2001) and play a role in directly into pre-existing CKFs (e.g., Chou et al., 1993; cellular stress responses (Liao et al., 1995; Liao et al., 1997; Bachant and Klymkowsky, 1996). The molecular interactions Omary et al., 1998; Gilbert et al., 2001; Marceau et al., 2001; of IF polypeptides are very strong, thus favouring the 4134 Journal of Cell Science 115 (21) filamentous over the soluble state by far. It takes saturated urea et al., 1991; Miller et al., 1993). One would expect that solutions to completely break up CKFs into their monomeric filaments are broken down and reassemble in various cellular subunits (Franke et al., 1983). In spite of these extreme domains to support ongoing cellular functions such as biochemical properties, CKs must be in an adjustable movement of vesicular carriers and organelles within the equilibrium between the soluble and filamentous state in vivo cytoplasm and migration of cells or their rearrangement within to allow processes that require a less rigid and more pliable a complex tissue. This type of plasticity should fulfil certain cytoskeleton. requirements: it must be rapid to respond immediately to Consequently, CKFs can not be viewed simply as rigid and dynamic requirements; it must be reversible to prevent immobile scaffoldings around which the cell body is arranged. interference with basic CK functions; and it must be restricted On the contrary, the arrangement and dynamic properties of the temporospatially to prevent catastrophic disruption of the IF CKF cytoskeleton must be in constant interplay with cellular cytoskeleton and interference with its basic housekeeping requirements. Probably the best examined physiologically functions. As a first step in the identification of such dynamic occurring process of major IF rearrangement takes place during principles of CK organisation, we now show by in vivo mitosis, when CKFs either disassemble completely into soluble fluorescence microscopy that the tyrosine phosphatase subunits and granular aggregates or collapse into cage-like inhibitor orthovanadate (OV) induces alterations in the CKF thick filament bundles (Franke et al., 1982; Lane et al., 1982; network that fulfil the above-mentioned requirements. These Jones et al., 1985; Kitajima et al., 1985; Tölle et al., 1987; observations give rise to the exciting possibility that transient Windoffer and Leube, 1999; Windoffer and Leube, 2001; CKF disruption is regulated through signaling pathways that Windoffer et al., 2002). Protein modification and altered involve phosphorylation. interactions with IF-associated proteins (IFAPs) are supposed to be major factors contributing to such CKF network remodeling. Accordingly, increased levels of phosphorylated Materials and Methods CK polypeptides were detected in mitotic cells (Celis et al., Construction of PLC cells stably expressing fusion protein 1983; Chou and Omary, 1993; Liao et al., 1997; Omary et al., HK18-YFP 1998) and also during meiosis (Klymkowsky et al., 1991). To generate a cDNA construct coding for a fluorescent CK18 chimera, ′ Phosphorylation may affect CK organization in a number of we amplified a 270 bp fragment of the 3 end of the coding region of different ways: it leads to a shift in the equilibrium between cDNA clone pHK18-P-7 (Bader et al., 1991) by polymerase chain reaction (PCR) with amplimers 99-16 5′-CTC AAC GGG ATC CTG the soluble and filamentous state toward the soluble form CTG CA-3′ and 99-17 5′-TTT GGT ACC CCA TGC CTC AGA ACT (Chou and Omary, 1993); it may directly induce filament TTG GTG T-3′. The BamHI/Asp718-cleaved PCR product was cloned disassembly as suggested by in vitro experiments (Yano et al., into pBluescript KS+ (Stratagene, La Jolla, CA) and complemented 1991); and it alters the interaction with IFAPs, as demonstrated by the 1 kb BamHI 5′-fragment of pHK18-P-7, thereby generating for the binding to the signaling 14-3-3 proteins, which act as clone pHK18∆stop. The EcoRI/Asp718 insert of this plasmid was solubility factors for CKs (Liao and Omary, 1996; Ku et al., transferred into vector pEYFP-N1 (Clontech Laboratories, Palo Alto, 1998). Furthermore, phosphorylation of the cytoskeletal CA). The resulting recombinant plasmid HK18-YFP1 confers crosslinker plectin may affect CK organisation, as has been neomycin resistance and codes for the hybrid HK18-YFP, which shown for plectin–lamin-B and plectin-vimentin interactions consists of complete human CK18 that is connected to the enhanced (Foisner et al., 1991; Foisner et al., 1996). yellow fluorescent protein by the short linker sequence GDPPVAT. The expression of this chimera is under the control of the CMV Among the non-filamentous assemblies of CKs, granular promoter. aggregates are of particular relevance for the understanding of To obtain cell lines stably expressing HK18-YFP, we used a disease pathologies. For example, during chronic liver disease modified calcium phosphate precipitation method (Leube et al., 1989). Mallory bodies are formed, which contain large amounts of Transfected cells were selected using geneticin sulphate (Invitrogen hyperphosphorylated CK polypeptides (Franke et al., 1979; Life Technologies, Karlsruhe, Germany). Surviving colonies were Cadrin and Martinoli, 1995; Stumptner et al., 2000). In this picked, transferred into MicrotestM Tissue Culture Plates (Becton case, phoshorylation itself may be the cause of aggregation Dickinson Labware, Franklin Lakes, NJ) and amplified for further (Yuan et al., 1998), possibly by preventing ubiquitin-dependent analysis. degradation (Ku and Omary, 2000; Coulombe and Omary, 2002). Granular aggregates are also a hallmark of various Cell culture genodermatoses (Anton-Lamprecht, 1983; Coulombe et al., AK13-1 cells expressing the chimera HK13-EGFP (Windoffer and 1991; Cadrin and Martinoli, 1995; Kobayashi et al., 1999). Leube, 1999) and hepatocellular-carcinoma-derived PLC cells stably Furthermore, granules are formed in vitro in response to expressing fusion protein HK18-YFP were grown in high glucose temperature stress or to drugs that affect the state of cellular DMEM (PAA Laboratories, Cölbe, Germany) at 37°C with 5% CO2. phosphorylation (Schliwa and Euteneuer, 1979; Shyy et al., In some instances, okadaic acid (Sigma, St. Louis, MO) was dissolved 1989; Falconer and Yeung, 1992; Kasahara et al., 1993; Liao in DMSO at 10 µg/ml and added to cells at final concentrations et al., 1995; Blankson et al., 1995; Toivola et al., 1997; Strnad between 0.1 µg/ml and 1 µg/ml. In other instances, sodium et al., 2001). Elucidation of the mechanisms of granule orthovanadate (OV; Aldrich Chemical Corporation, Milwaukee, WI) formation may reveal ways to influence their formation and was dissolved in distilled water and stored as a 1 M stock. It was either used directly or incubated with H2O2 prior to usage to generate thereby positively affect disease outcome. pervanadate (Feng et al., 1999). In some cases, especially for time- Very little is known about the molecular principles that lapse fluorescence microscopy, phenol-red-free Hanks medium was determine the dynamic CKF organisation in interphase cells used (cf. Strnad et al., 2001). To enrich for mitotic cells, cultures were beyond the continuous and non-selective exchange of subunits treated for 6 hours with demecolcine (Sigma) at a final concentration throughout the entire CKF network (Franke et al., 1984; Miller of 20 µM. Dynamic cytokeratin network organisation 4135

Immunofluorescence microscopy epifluorescence microscopy using inverse optics from Olympus Cells grown on 18 mm glass coverslips to the desired density were (Hamburg, Germany) and an attached IMAGO slow scan CCD fixed with pre-cooled methanol (5 minutes, –20°C) and acetone (15 camera. The microscope was placed in a heated chamber (37°C). The seconds, –20°C), and mounted either directly in elvanol or subjected whole system was controlled by TILLvisION software. Excitation to indirect immunofluorescence prior to embedding as described was adjusted with the monochromator to 496 nm for the detection of previously (Windoffer and Leube, 1999; Strnad et al., 2001). EGFP and to 498 nm for EYFP. Image sequences were imported into Fluorescence was viewed in an epifluorescence microscope Image-Pro Plus 4.5 (Media Cybernetics) and converted into movies (Axiophot, Carl Zeiss, Jena, Germany) and was recorded with a digital (available at jcs.biologists.org/supplemental). Photoshop software camera (Hamamatsu 4742-95, Hamamatsu, Herrsching, Germany). (Adobe Photoshop 5.0) was used to edit single pictures and to Murine monoclonal antibodies were used for detection of plectin assemble figures. (clone 10F6; kindly provided by Gerhard Wiche, Biocenter, Vienna, Austria) (Foisner et al., 1994) and α-tubulin (Amersham Pharmacia Biotech, Freiburg, Germany). Monoclonal CK epitope antibodies Gel electrophoresis and immunoblotting were generously provided by Bishr Omary and Nam-On Ku (Stanford High salt pellet fractions containing enriched CKs and corresponding University, Palo Alto, CA), reacting with CKs 8 and 18 (L2A1) (Chou supernatant fractions were prepared in the continued presence of OV et al., 1993), phospho-S73 of CK8 (LJ4) (Liao et al., 1997), phospho- (5 mM) prior to one-dimensional SDS PAGE or two-dimensional gel S431 of CK8 (5B3) (Ku and Omary, 1997) and phospho-S33 of CK18 electrophoresis employing isoelectric focusing in the first dimension (IB4) (Ku et al., 1998). Polyclonal antibodies from rabbits against using established procedures (Achtstaetter et al., 1986). Separated CK5 were from Regina Reichelt and Thomas Magin (Department of polypeptides were either stained with Coomassie Brilliant Blue Biochemistry, Bonn University, Germany) (Peters et al., 2001), (Serva, Heidelberg, Germany) or were transferred onto nitrocellulose against plectin from Harald Herrmann (German Cancer Research membranes for immunoblotting. In addition to the above-mentioned Center, Heidelberg, Germany) (Schröder et al., 1999). For detection CK antibodies monoclonal antibodies against phospho-tyrosine of protein 14-3-3, isoform-ζ-specific and broad-reactive rabbit epitopes (P-Tyr-100; New England Biolabs GmbH, Frankfurt, antibodies (antibodies sc-1019 and sc-629, respectively) were Germany) and CK13 (Ks13.1, Progen Biotechnics, Heidelberg, purchased from Santa Cruz Biotechnology (Santa Cruz, CA). To Germany) were used. Detection of bound antibodies was visualise bound primary antibodies, Texas-Red-conjugated goat anti- accomplished with horseradish-peroxidase-coupled secondary rabbit antibodies (Jackson ImmunoResearch Laboratories, West antibodies (Jackson ImmunoResearch Laboratories) and an enhanced Grove, PA) and Cy3-conjugated goat anti-mouse IgGs (Rockland chemiluminescence system (Amersham Biosciences Europe, Laboratories, Gilbertsville, PA) were used. Freiburg, Germany). For actin staining, cells were briefly washed in PBS prior to incubation with Texas-Red-coupled phalloidin (Molecular Probes, Eugene, OR) at room temperature for 30 minutes, which was followed Results by washing in PBS (10 minutes) and distilled water (2 minutes) before mounting in elvanol. Orthovanadate induces multifocal and reversible For statistic analysis, digital images were imported into Image-Pro cytokeratin filament network disruption and granule Plus 4.5 (Media Cybernetics, Silver Spring, CA) to calculate formation Pearson’s correlation coefficients. The resulting data were further To examine the effect of the tyrosine phosphatase inhibitor analysed for statistically significant differences between groups with orthovanadate (OV) on CKF network organization, vulva- two-tailed Wilcoxon-W-test using SPSS software (version 9; SPSS carcinoma-derived A-431 subclone AK13-1 stably Incorporation, Chicago, IL). The determined Pearson’s coefficients expressing fluorescent human CK13 chimera HK13-EGFP were transferred to SigmaPlot 2001 (Jandel Scientific GmbH, Erkrath, was chosen, because it is well suited for multidimensional Germany) to prepare Whisker-box-blots. monitoring of CKs in living cells (Windoffer and Leube, 1999; Windoffer and Leube, 2001; Strnad et al., 2001). Electron microscopy Remarkably, most of the CKF network disappeared within Cells were grown on glass slides and washed briefly in PBS prior to less than 10 minutes of addition of OV, and the entire a 2 hour fixation in a freshly prepared solution of 2% (w/v) cytoplasm became filled with numerous small granules. After paraformaldehyde and 2.5% (w/v) glutaraldehyde in phosphate buffer 2 minutes, the majority of emerging granules was in close (0.05 M Na2HPO4, 0.05 M NaH2PO4). Subsequently, cells were apposition to or in direct continuity with remaining filaments, washed three times for 5 minutes in phosphate buffer containing 0.1 connecting them like pearls on a string (Fig. 1B, Fig. 2A,B). M sucrose and were postfixed for 30 minutes with osmium tetroxide At this stage, cells were still outspread with only minor [2% (w/v) in PBS]. After two 5 minute washes in distilled water, cells were dehydrated in an ascending ethanol series. Embedding was done retractions at the cell surface as judged by phase-contrast in Epon 812 (Serva, Heidelberg, Germany). Ultrathin sections were microscopy (data not shown). Chromatin staining with stained with 8% (w/v) uranyl acetate for 10 minutes and contrasted Hoechst 33258 was indistinguishable from that of untreated with lead citrate for 5 minutes. Sections were viewed in an EM 906 cells (data not shown). Subsequently, fragmented structures (Zeiss, Oberkochen, Germany). Immunoelectron microscopy was were occasionally observed (Fig. 2C), and after 10 minutes done as described previously (Windoffer and Leube, 1999; Strnad et of OV treatment, practically all cells were rounded and CKFs al., 2001) using antibodies against green fluorescent protein that had completely disappeared (Fig. 1C). crossreact with enhanced green fluorescent protein (Molecular Probes, In the corresponding electron micrographs, bundled Eugene, OR) in combination with gold-labelled secondary antibodies filaments with local thickenings appeared (Fig. 2E). Some of and the silver amplification technique. the thickenings contained an amorphous core, probably representing an early state of non-filamentous granular Time-lapse fluorescence microscopy structures (+ in Fig. 2E). Many of the emerging spheroidal To view living cells, Petri dishes with glass bottoms (MatTek granules were in direct continuity with residual filaments (Fig. Corporation, Ashland, MA) were used. Images were recorded by 2E and inset). Practically all filaments were gone in the final 4136 Journal of Cell Science 115 (21)

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.

reversible, OV was washed out after 10 minutes. Indeed, cells restored their typical outspread morphology within 50 minutes of removal of the drug. Remarkably, all granules were gone by this time, and an extensive CKF network had been re-established (Fig. 1D). This network was finer than the typical interphase web, and the morphological appearance remained practically unchanged for the next 5 hours (data not shown). To demonstrate that the changes in HK13-EGFP-containing filaments reflected the behaviour of the entire CKF system, the endogenous network was examined by indirect immunofluorescence in wild-type A-431 cells and AK13-1 cells. Direct comparison of CK5 immunofluorescence with HK13-EGFP fluorescence before OV-treatment, 2 and 10 minutes after addition of the drug and 50 minutes after Fig. 1. Double fluorescence microscopy of methanol/acetone-fixed washout showed that both patterns were practically identical AK13-1 cells to detect the distribution of the CK13-EGFP chimera (Fig. 1A′-D′). Similar results were also obtained, when HK13-EGFP (A-D) together with human CK5 using polyclonal antibodies against CKs 8 and 18 were used (data not shown). rabbit antibodies (A′-D′) before (A,A′) during (B,B′,C,C′) and 50 ′ In most instances, however, staining of granules with CK minutes after a 10 minute treatment with 5 mM OV (D,D ). Note the antibodies was considerably weaker than the corresponding progressive loss of CKFs during OV incubation accompanied by formation of granules throughout the cytoplasm, which remain in HK13-EGFP fluorescence. In particular, large granules were close association with residual filaments. Both processes are labelled only on their surface by indirect fluorescence reversed upon removal of the drug, yielding a fine CKF microscopy, indicating that epitopes within granules were cytoskeleton. Bars, 10 µm. either altered or, more likely, not accessible to the antibodies, as it was also evident from immunoelectron microscopy (Fig. 2G,H). stages and, instead, multiple non-filamentous granules ranging The reaction of all cells in a given experiment was in diameter from less than 200 nm to 500 nm were spread exceptionally uniform, and the kinetics of granule formation throughout the cytoplasm without apparent linkage (Fig. 2F). were similar for OV concentrations between 2 and 10 mM. Immunoelectron microscopy using antibodies against EGFP However, considerably reduced OV sensitivity was often noted confirmed that the dense bundles with local enlargements and in cells grown in phenol-red-free Hanks medium in comparison the newly formed aggregated material contained HK13-EGFP to those maintained in high glucose DMEM [for medium (Fig. 2G,H). dependency of OV see also (Huyer et al., 1997)]. Furthermore, To examine whether these drastic alterations were OV activation by H2O2 did not affect the observed pattern of Dynamic cytokeratin network organisation 4137

CKF reorganisation, although approximately 10-times lower Orthovanadate effects on the cytokeratin filament concentrations of pervanadate were sufficient to cause network show no apparent correlation to alterations in comparable alterations. For the following experiments the non- the microtubule and actin system activated form of OV was used. In some instances, cell Next, we examined whether OV-induced CKF network changes morphology normalised even without removal of the drug, correlate with alterations of other cytoskeletal components. probably indicating inactivation of OV and/or acquisition of After 2 and 10 minutes of OV treatment, the microtubule insensitivity to the drug (see also Huyer et al., 1997). network was still visible (Fig. 3B′,C′; see also arrowheads in Concurrently, granules disappeared to a large extent and CKFs Fig. 2E). However, the number of microtubules was slightly were restored. reduced, and microtubules were distributed differently, 4138 Journal of Cell Science 115 (21)

Fig. 3. Fluorescence microscopy of methanol/acetone-fixed AK 13-1 cells detecting the chimera HK13-EGFP (A-H) together with microtubules using anti α-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. sometimes in proximity to CK granules (Fig. 2E, Fig. 3B,B′), mediates interactions among all three major cytoskeletal which probably reflected only the cell shape change and not a filament networks (Foisner, 1997; Wiche, 1998), was specific association. After 2 minutes of OV incubation, an examined next. Plectin immunofluorescence partially co- increase in actin stress fibers was noted (Fig. 3F′). Later on, distributed with HK13-EGFP fluorescence in interphase actin was further concentrated in the cell cortex (Fig. 3G′). After AK13-1 cells when either monoclonal (Fig. 4A,A′) or washout of OV, both microtubules and the actin system were polyclonal antibodies were used (data not shown). A largely restored within 50 minutes (Fig. 3D′,H′). Taken significant increase in colocalisation was apparent after 2 together, these observations show that alterations in these minutes of OV addition (Fig. 4B,B′) and reached near identity systems do not correlate with CKF network remodeling. after 10 minutes (Fig. 4C,C′). After removal of OV, newly formed CKFs still showed a somewhat increased co- distribution with plectin (data not shown). To quantify the Orthovanadate-induced changes in plectin distribution changes in plectin-CK co-distribution, Pearson’s correlation reflect altered cytokeratin filament network organisation coefficients were determined between HK13-EGFP and resemble alterations observed during mitosis fluorescence and plectin immunofluorescence in untreated The distribution of the IF-binding protein plectin, which cells, in cells with completely disrupted CKF networks (10 Dynamic cytokeratin network organisation 4139

Fig. 4. Fluorescence microscopy depicting the distribution of HK13-EGFP (A-K) together with plectin (A′-E′; monoclonal antibodies 10F6) or 14-3-3 ζ (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 ζ distribution exhibiting the reverse behavior. Bars, 10 µm. 4140 Journal of Cell Science 115 (21)

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 Fig. 5. Whisker-box plots showing colocalisation between HK13- of 50 mM OV (C). Note the loss of filaments and the formation of EGFP fluorescence and plectin immunostaining in AK13-1 cells rods and granules in C. D shows inverse contrast images at high using Pearson’s correlation coefficient. For each group, eight typical magnification that were taken from a live cell recording (Movie 1; double-labelled cells were analysed. The top and bottom of each box recording intervals 1 second) demonstrating details of OV-induced delineate the upper and lower 25 percentiles, respectively, the center CKF-disruption. Note the straightening of CKFs (arrows) prior to line demarcates the median and the dots depict the minimum and fragmentation into rods and granules. Bars, 10 µm in (A-C) and 1 maximum values. UC, untreated interphase cells; OV, cells treated µm in (D). 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. okadaic acid treatment were positive for 14-3-3 proteins (Fig. 4I,I′), IFAPs that preferentially associate with soluble and phosphorylated CK subunits (Liao and Omary, 1996). By minutes of OV treatment) and in partially recovered cells 50 contrast, neither OV-induced granules nor those generated minutes after removal of OV. For each situation, eight typical during mitosis contained significant amounts of 14-3-3 regions were chosen on the basis of DNA staining without proteins (Fig. 4G,G′,H,H′). Taken together, our results show prior knowledge of the plectin distribution. The results (Fig. that aggregates formed during okadaic acid treatment differ 5) were fully in support of the visual evaluation, revealing a from those formed during OV incubation and mitosis. significant increase in colocalisation between plectin and HK13-EGFP both after 10 minutes of OV treatment and after an additional 50 minutes recovery period in comparison to Cytokeratin granules are generated directly from untreated cells with a P value of <0.005. Furthermore, co- cytokeratin filaments and cytokeratin filament fragments distribution was slightly lower after recovery in comparison to during orthovanadate treatment the time of maximum CKF disruption, although the difference Time-lapse fluorescence microscopy was performed in AK13- was statistically insignificant (P=0.08). 1 cells to find out how CK granules are formed during OV To find out whether the increased colocalisation of plectin treatment. Using this method, small granules were seen to and CKs is a common feature of CKF disruption, we examined rapidly emerge from filaments (Fig. 6A). Further details of this two other situations in which granular aggregates are formed. process were difficult to image owing to its speed and the First, cells were treated with the serine/threonine-phosphatase simultaneous rounding of cells, which resulted in focal shifts inhibitor okadaic acid (see also Strnad et al., 2001). In this of fluorescent structures. For better resolution we therefore instance, granules did not significantly co-distribute with established hepatocellular-carcinoma-derived PLC cells plectin at any time point during CKF disruption (Fig. 4D,D′), expressing fluorescent CKFs. The cytoplasm of these cells is which was also evident from Pearson’s coefficient analysis much more outspread than it is in A-431 cells, and the CKF (Fig. 5). The P value of the analysis for okadaic-acid-treated network has a larger mesh size with thicker appearing and untreated control cells was >0.8, suggesting no significant filaments. A HK18-YFP chimera was stably introduced into difference between both groups, whereas the P value of the PLC cells. The fluorescent chimeras were incorporated into a Pearson’s coefficient analysis for okadaic-acid-treated cells typical CKF network (Fig. 6B) and co-distributed with the and cells incubated for 10 minutes with OV was <0.0002, endogenous CKs (data not shown). Although PLC cells were demonstrating the significant difference between both granule less sensitive to OV than A-431 cells, similar alterations were types. Second, double fluorescence microscopy was performed induced (Fig. 6C). The time until filament breakdown began during mitosis when granular and rod-like aggregates are was usually longer in PLC than in A-431 cells even when high formed and additional strong diffuse fluorescence occurs as a concentrations of OV were used. Yet, the breakdown itself result of increased soluble subunits in the cytoplasm of AK13- was remarkably fast once it had started and occurred 1 cells (Windoffer and Leube, 2001). Despite the obscuring homogeneously throughout the entire cell. High magnification effect of the diffuse HK13-EGFP staining, a strong correlation images of distinct CKF bundles showed that they fragmented was observed between fluorescent HK13-EGFP aggregates and and formed small rodlets and/or granules (Fig. 6D). plectin immunofluorescence during mitosis (Fig. 4E,E′). This Interestingly, CKFs straightened prior to disruption (arrows was also confirmed by Pearson’s coefficient analysis (Fig. 5), Fig. 6D). The dynamic aspects of these processes are best which produced a P value of <0.0007 for comparison with appreciated in Movie 1 (available at jcs.biologists.org/ untreated cells. supplemental), in which precursor-product relationships are Interestingly, the plectin-negative granules formed during unambiguously resolved. Dynamic cytokeratin network organisation 4141

Orthovanadate-induced cytokeratin filament network lapse fluorescence imaging suggested that the granules formed reorganisation is rapidly reversible as local thickenings within or next to CKFs and that they were In a few instances, OV treatment resulted only in incomplete re-integrated directly into the original filaments afterwards disassembly of the CKF network. In this case, it was possible (arrows in Fig. 7). After re-integration, the CKF network to trace small CK granules, which disappeared again after a looked similar to that prior to treatment, although the cells were short time (usually within less than 30 minutes; Fig. 7). Time- still slightly contracted (see Fig. 7, 38 minutes). Movie 4142 Journal of Cell Science 115 (21) sequences taken at high frequency provided further evidence of CK granule formation. Even when most of the CKF network for the reversible nature of OV-induced granule formation by was disrupted and the entire cytoplasm was filled with small direct transformation into and from CKFs (Movie 2; available granules, fast reformation of an extended CKF network took at jcs.biologists.org/supplemental). Furthermore, the movies place after OV washout (Fig. 8 and corresponding Movies 3,4, demonstrated that the granules stayed in close apposition to the available at jcs.biologists.org/supplemental). Although no filaments during the entire process, suggesting a remaining continuous connection between such granules was noticeable, connection. Movie 2 also revealed peripheral CKFs that they still appeared to be anchored either to CKFs that were were most probably anchored to desmosomal contact sites below the detection limit and/or to other parts of the (arrowheads in Fig. 7; see also arrowheads in Fig. 6A). Loss cytoskeleton. The CK granules completely disappeared within of cell adhesion upon OV treatment resulted in retraction of 25 minutes of removal of OV, and a very delicate but extensive these filaments, which appeared to remain in contact with the and dense network was re-established (Fig. 8). Reformation of plasma membrane. the network was not restricted to certain cellular subdomains To investigate the re-formation of a CKF network from OV- but occurred at multiple sites throughout the cytoplasm. High induced granules in a more controlled fashion, OV was washed magnification revealed further details of this process (Movie out after short incubation periods prior to time-lapse 4). Granules were seen to elongate and to fuse occasionally. fluorescence microscopy. In general, the same phenomena They decreased in size and disappeared gradually whereas thin were seen as in cells with spontaneously occurring reversion filaments were formed, often extending from the vanishing

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. Dynamic cytokeratin network organisation 4143 granules. The first visible filaments were just above The solubility and phosphorylation of the majority of background fluorescence, subsequently gaining intensity and cytokeratin polypeptides is not significantly altered in generating a finely woven network. This newly formed network orthovanadate-treated cells differed in appearance from the coarser network in most To examine if and to what degree CKs were altered by OV, interphase cells (e.g., Fig. 1A, Fig. 3A,E, Fig. 4A,F). we looked for biochemical changes in cells with completely Furthermore, the network was remarkably homogeneous, disrupted CKF networks, as assessed by fluorescence lacking, most notably, the thick perinuclear filament bundles microscopy. We found no significant increase in CK and prominent desmosome-anchored filaments. However, the solubility in OV-treated cells in comparison with untreated peripheral part of the newly formed CKF network exhibited the cells (Fig. 9A). This was in contrast to elevated levels of typical inward-directed movement of CK fluorescence soluble CKs in okadaic-acid-treated and enriched mitotic [compare Movie 3 with movies shown in (Windoffer and cells (Fig. 9A). Next, two-dimensional gel electrophoresis Leube, 1999)]. was performed to search for altered patterns of CK

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. 4144 Journal of Cell Science 115 (21)

Fig. 9. Gel electrophoretic analysis of CKs in AK13-1 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 Ks13.1 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. modification. No major differences between OV-treated and which was, however, below the detection limit. In fact, untreated cells were apparent, although some minor the observed fragmentation of CKFs suggests that the differences were seen, most notably for CK 5 (Fig. 9B,C). modification of a very small proportion of CK polypeptides in Furthermore, commercially available phospho-tyrosine restricted hot spots would be sufficient to disrupt the CKF antibodies did not reveal any significant reactivity of network. Furthermore, phosphorylation of CKs appears to be cytoskeletal fractions before or after OV incubation (data not the major principle by which CKF network organisation is shown). Similarly, antibodies reacting with specific phospho- regulated in vivo (e.g. Celis et al., 1993; Klymkowsky et al., serine epitopes of CKs 8 and 18 did not pick up increased 1991; Chou and Omary, 1993; Stumptner et al., 2000). On the phosphorylation, although some of the sites were clearly other hand, alternative explanations need to be considered elevated after okadaic acid treatment (Fig. 9D). These given the non-specific nature of OV, which may exert its observations suggest that the percentage of modified CKs in effects indirectly via IFAPs and/or other cytoskeletal OV-treated cells is either below the detection limit of the components. Either way, the examination of OV-treated cells methods used so far and/or that other factors are responsible revealed physiologically relevant principles of CK dynamics, for the observed effects. which are directly or indirectly regulated by phosphorylation. The speed of the observed granule formation and the reversibility of this process are features that would be needed Discussion for rapid adaptation of cells not only to gross cell shape Orthovanadate-induced changes in cytokeratin changes as they occur during mitosis or migration but also to organisation serve as a paradigm for phosphorylation- local alterations that take place throughout the lifetime of each dependent cytokeratin filament network regulation interphase cell. Furthermore, epidermal growth factor and We detected, for the first time, reversible CKF network stress-activated protein kinases such as p38, which have been breakdown in living cells as a result of transient treatment with shown to affect CK phosphorylation (Aoyagi et al., 1985; the tyrosine phosphatase inhibitor OV. Pervanadate induced Baribault et al., 1989; Liao et al., 1997; Feng et al., 1999; Ku the same reversible process with identical kinetics, albeit at et al., 2002), are potential and well suited regulators of lower concentrations, suggesting that it acts in a similar restricted CKF disassembly and assembly. The dynamic on/off fashion. We found that the phosphorylation and solubility of property of phosphorylation of CK 8 S73 and of tyrosine the majority of CKs was not altered at times of complete residues of CKs 8/19 by such enzymes (Feng et al., 1999; Ku CK network breakdown. Yet, using considerably longer et al., 2002) adds further support to the suitability of these incubation periods of 60-90 minutes, it has been demonstrated mechanisms. In this way, local phosphorylation of CKs and/or that pervanadate leads to an increase in tyrosine associated mediators could result in a disassembly that would phsophorylation, serine phosphorylation and solubility of CKs be reversed after removal of the phosphate group, thereby 8 and 19 (Feng et al., 1999). It therefore remains a possibility inducing temporally and spatially restricted remodeling of the that a minor fraction of CKs was also modified in our cells, CKF cytoskeleton. Dynamic cytokeratin network organisation 4145 Orthovanadate-induced cytokeratin filament remodeling Plectin is important for cytokeratin filament network re- is similar to that occurring during mitosis but is formation profoundly different from that mediated by okadaic acid We were particularly intrigued by the observed association of The effect of various modulators of phosphorylation on the plectin with OV-induced CK granules. Among the various dynamics and organization of the CKF cytoskeleton has been linker proteins of the plakin type that connect the cytoskeletal the subject of many studies (Baribault et al., 1989; Eckert and filament systems to each other and to certain plasma membrane Yeagle, 1990; Cadrin et al., 1992; Falconer and Yeung, 1992; domains (Ruhrberg and Watt, 1997; Fuchs and Karakesisoglou, Ohta et al., 1992; Deery, 1993; Kasahara et al., 1993; 2001), plectin is certainly the most prominent crosslinker for Yatsunami et al., 1993; Baricault et al., 1994; Blankson et al., the IF system (reviewed in Foisner, 1997; Wiche, 1998). 1995; Toivola et al., 1997; Toivola et al., 1998; Yuan et al., Interestingly, plectin affects assembly properties of both 1998; Feng et al., 1999; Paramio, 1999; Sanhai et al., 1999; unassembled and assembled IF polypeptides, including CKs, Negron and Eckert, 2000; Strnad et al., 2001). One of the best in a complex fashion (Steinböck et al., 2000). Furthermore, examined drugs in this context is the serine/threonine plectin is subject to dynamic phosphorylation, most notably phosphatase inhibitor okadaic acid, which induces complete during M phase, when increased p34cdc2 kinase-mediated disruption of the CKF system and results in formation of phosphorylation of plectin is accompanied by its redistribution granular aggregates (Kasahara et al., 1993; Yatsunami et al., from a mostly filament-associated to a diffuse state (Foisner et 1993; Chou and Omary, 1994; Blankson et al., 1995; Strnad et al., 1996). The observed colocalisation of plectin with OV- al., 2001). The effects of okadaic acid, however, differ in induced CK granules was specific, since other CK-associated several respects from those induced by OV as reported in this proteins, such as the desmosomal proteins desmoplakin and communication. The okadaic-acid-induced disruption takes plakophilin (data not shown) and the signalling molecules of several hours to complete, starting only after a lag period of 1- the 14-3-3 type did not co-distribute. It therefore appears 2 hours in AK13-1 cells, and cells did not recover for several reasonable to assume that the particular composition of hours after removal of the drug (Strnad et al., 2001) (see also granular CK aggregates determines their dynamic properties. Lee et al., 1992) (P.S., R.W. and R.E.L., unpublished). Hence, given the compositional differences between okadaic- Furthermore, increased levels of soluble CKs and CK phospho- acid-induced and OV-induced granules we propose that plectin epitopes were readily identified after okadaic acid treatment defines, in a yet unknown manner, CK aggregates that can be but not after OV. In addition, okadaic-acid-induced granules rapidly re-integrated into filaments. A possibility is that plectin were, on the average, more than twice the size of OV granules maintains anchorage of granules to the cytoskeleton, since [compare Fig. 2E-H with (Strnad et al., 2001)]. They also differ plectin-positive granules were rather immobile in contrast to compositionally by the presence of 14-3-3 proteins and the those seen during okadaic-acid treatment (Strnad et al., 2001). absence of plectin (this study). Finally, in okadaic-acid-treated On the other hand, loss of plectin and association with 14-3-3 cells, CKFs disappear first in the cell periphery and only later proteins abolishes the capacity of CK aggregates to re-establish in the perinuclear region (Strnad et al., 2001). Although a CKF system. okadaic acid is also known to induce p38 (Westermarck et al., 1998; Chen et al., 2000) and to affect p38-mediated phosphorylation of IFs and IFAPs (Cheng and Lai, 1998; Chen Cytokeratin network formation is determined by two et al., 2000; Ku et al., 2002), it must act differently from OV. mechanistically different pathways We recently proposed that the effect of okadaic acid is mainly We have shown here that the CKF network recuperates after caused by prevention of integration of soluble CK subunits into OV-induced breakdown within a short time. However, two CKFs, which occurs preferentially in the cell periphery (Strnad alternative modes of CKF re-formation were noted. Most et al., 2001). The observed colocalisation of CK aggregates frequently, multiple cytoplasmic CK granules were seen to with 14-3-3 protein, which may keep CKs ‘soluble, that is, vanish by integrating into a fine filamentous network. This non-filamentous, further supports this notion (Liao et al., multifocal CKF assembly mechanism is highly reminiscent 1997). This also explains the centripetal decrease in filaments of experiments in which CKs were introduced into epithelial in okadaic-acid-treated cells, where cortical filament and non-epithelial cells by microinjection of epidermal poly- reformation (Windoffer and Leube, 1999; Windoffer and A+-RNA or by transfection with CK cDNA-constructs, in Leube, 2001) appears to be blocked, thereby resulting in a which multiple sites were shown to be involved in CKF filament-free peripheral cytoplasm while perinuclear filament network formation (Kreis et al., 1983; Franke et al., 1984; bundles are still intact. However, this process is rather slow and Magin et al., 1990; Bader et al., 1991). The relevance of this therefore not sufficient to account for the rapid CKF disruption mechanism was further strengthened by the experiments of observed in OV-treated cells and at the beginning of mitosis Miller et al. that demonstrated that microinjected CK (Windoffer and Leube, 2001). The morphological similarities polypeptides first formed granules together with their of mitotic CKF breakdown to OV-induced changes are striking, endogenously expressed partner polypeptides and integrated since small, filament-associated granules were found in both subsequently at multiple sites into the IF cytoskeleton of instances during the rapid disruption process which takes less epithelial cells (Miller et al., 1991; Miller et al., 1993). than 10 minutes (Windoffer and Leube, 2001). Furthermore, Their images are practically indistinguishable from our we have shown here that mitotic granules contain, similar to photomicrographs of OV-treated cells [compare, for example, OV-induced granules, plectin and lack 14-3-3 protein. Whether Fig. 2A-C and Fig. 7 with Fig. 7 of (Miller et al., 1993)]. the underlying molecular mechanisms of granule formation Furthermore, ultrastructural analyses showed in both and disassembly are identical in both situations remains to be instances that the granules were composed of non- determined. filamentous material and were in direct continuity with IF 4146 Journal of Cell Science 115 (21) bundles. Finally, the speed of integration of granular material and chromatographic techniques and their identification by immunoblotting. into filaments is comparable. Methods Enzymol. 134, 355-371. The other mode of CKF reformation after OV treatment was Anton-Lamprecht, I. (1983). Genetically induced abnormalities of epidermal differentiation and ultrastructure in ichthyoses and epidermolysis: observed only in a minority of cells and will be presented in pathogenesis, heterogeneity, fetal manifestation, and prenatal diagnosis. J. detail elsewhere. In this case, novel CKFs were exclusively Invest. Dermatol. 81, (Suppl. 1), 149s-156s. detected in the cell periphery, which was similar to the recently Aoyagi, T., Suya, H., Umeda, K., Kato, N., Nemoto, O., Kobayashi, H. and described CKF network reformation after mitosis in AK13-1 Miura, Y. (1985). Epidermal growth factor stimulates tyrosine phoshorylation of pig epidermal fibrous keratin. J. Invest. Dermatol. 84, cells (Windoffer and Leube, 2001). This cortex-restricted mode 118-121. is significantly slower than multifocal remodeling. Apparently, Bachant, J. B. and Klymkowsky, M. W. (1996). A nontetrameric species is factors that are needed for successful cytoplasmic CKF network the major soluble form of keratin in Xenopus oocytes and rabbit reticulocyte formation were inactivated in some OV-treated cells and are lysates. J. Cell Biol. 132, 153-165. lacking after mitosis. Anchorage of CK granules to the Bader, B. L., Magin, T. M., Freudenmann, M., Stumpp, S. and Franke, W. W. (1991). Intermediate filaments formed de novo from tail-less cytoskeleton either by direct linkage to residual CKFs that are cytokeratins in the cytoplasm and in the nucleus. J. Cell Biol. 115, 1293- below the detection limit or to other, yet unknown, cytoskeletal 1307. components may be necessary for CKF network formation. In Baribault, H., Blouin, R., Bourgon, L. and Marceau, N. (1989). Epidermal support, cells with multiple cytoplasmic CKF-forming foci growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-kD cytokeratin before filament reorganizaton and DNA synthesis. J. Cell presented granules with low mobility throughout the cytoplasm. Biol. 109, 1665-1676. By contrast, extremely mobile granules that were observed in Baricault, L., de Nechaud, B., Sapin, C., Codogno, P., Denoulet, P. and the central cytoplasm during mitosis (Windoffer and Leube, Trugnan, G. (1994). The network organization and the phosphorylation of 2001) and sometimes after OV treatment (data not shown) lost cytokeratins are concomitantly modified by forskolin in the enterocyte-like the capacity for multifocal CKF network reformation and were differentiated Caco-2 cell line. J. Cell Sci. 107, 2909-2918. Bauman, P. A., Dalton, W. S., Anderson, J. M. and Cress, A. E. (1994). only able to contribute to cortical CKF formation. We therefore Expression of cytokeratin confers multiple drug resistance. Proc. Natl. Acad. propose that anchorage of CK granules in the cytoplasm, which Sci. USA 91, 5311-5314. may be mediated by plectin, is a prerequisite for successful Blankson, H., Holen, I. and Seglen, P. O. (1995). Disruption of the integration into a filamentous network. Probably cortex- cytokeratin cytoskeleton and inhibition of hepatocytic autophagy by okadaic acid. Exp. Cell Res. 218, 522-530. dependent and multifocal cytoplasmic CK-reorganization occur Cadrin, M. and Martinoli, M. G. (1995). Alterations of intermediate simultaneously but fulfil different functions. The multifocal filaments in various histopathological conditions. Biochem. Cell Biol. 73, principle would be expected to contribute to local and rapid 627-634. changes of the cytoskeleton and can be induced by specific Cadrin, M., McFarlane-Anderson, N., Aasheim, L. H., Kawahara, H., requirements in a temporospatially restricted fashion. Any out Franks, D. J., Marceau, N. and French, S. W. (1992). Differential phosphorylation of CK8 and CK18 by 12-O-tetradecanoyl-phorbol-13- of balance situation, such as the elevated presence of CKF acetate in primary cultures of mouse hepatocytes. Cell. Signal. 4, 715-722. polypeptides in cells that were microinjected with polypeptides Celis, J. E., Larsen, P. M., Fey, S. J. and Celis, A. (1983). Phosphorylation or mRNA (Miller et al., 1991; Miller et al., 1993; Franke et al., of keratin and vimentin polypeptides in normal and transformed mitotic 1984) or the OV-induced CKF-disruption, allows the detection human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis. J. Cell Biol. 97, 1429-1434. of this mechanism that is otherwise difficult to see in sessile Chen, K. D., Lai, M. T., Cho, J. H., Chen, L. Y. and Lai, Y. K. (2000). epithelial cell assemblies. The cortical principle, by contrast, Activation of p38 mitogen-activated protein kinase and mitochondrial Ca2+- may be a basic housekeeping function of epithelial cells, mediated oxidative stress are essential for the enhanced expression of grp78 sustaining the continuous CKF network replacement. induced by the protein phosphatase inhibitors okadaic acid and calyculin A. J. Cell. Biochem. 76, 585-595. Accordingly, a continuous inward-directed movement of CK Cheng, T. J. and Lai, Y. K. (1998). Identification of mitogen-activated protein fluorescence has been reported in living epithelial cells kinase-activated protein kinase-2 as a vimentin kinase activated by okadaic (Windoffer and Leube, 1999; Yoon et al., 2001). This system acid in 9L rat brain tumor cells. J. Cell. Biochem. 71, 169-181. would also be invoked in situations of complete disruption of Chou, C. F. and Omary, M. B. (1993). Mitotic arrest-associated enhancement the CKF network, because its associated structures are able to of O-linked glycosylation and phosphorylation of human keratins 8 and 18. J. Biol. Chem. 268, 4465-4472. initiate de novo filament formation as it occurs after mitosis and Chou, C. F. and Omary, M. B. (1994). Mitotic arrest with anti-microtubule after extended OV treatment (for details, see Windoffer and agents or okadaic acid is associated with increased glycoprotein terminal Leube, 2001). GlcNAcs. J. Cell Sci. 107, 1833-1843. Chou, C. F., Riopel, C. L., Rott, L. S. and Omary, M. B. (1993). A The expert technical help of Antje Leibold, Sabine Thomas and significant soluble keratin fraction in ‘simple’ epithelial cells. Lack of an Ursula Wilhelm is gratefully acknowledged. The authors thank apparent phosphorylation and glycosylation role in keratin solubility. J. Cell Sci. 105, 433-444. Gerhard Wiche (Vienna Biocenter, Vienna, Austria), Harald Coulombe, P. A. and Omary, M. B. 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