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Dynamic Cytokeratin Network Organisation 4135 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
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