The keratin-filament cycle of assembly and disassembly

Anne Klsch 1 Reinhard Windoffer 1 Thomas Wrflinger 0 Til Aach 0 Rudolf E. Leube 1 0 Institute of Imaging and Computer Vision, RWTH Aachen University , 52056 Aachen , Germany 1 Institute of Molecular and Cellular Anatomy, RWTH Aachen University , 52074 Aachen , Germany Summary Continuous and regulated remodelling of the cytoskeleton is crucial for many basic cell functions. In contrast to actin filaments and microtubules, it is not understood how this is accomplished for the third major cytoskeletal filament system, which consists of intermediate-filament polypeptides. Using time-lapse fluorescence microscopy of living interphase cells, in combination with photobleaching, photoactivation and quantitative fluorescence measurements, we observed that epithelial keratin intermediate filaments constantly release non-filamentous subunits, which are reused in the cell periphery for filament assembly. This cycle is independent of protein biosynthesis. The different stages of the cycle occur in defined cellular subdomains: assembly takes place in the cell periphery and newly formed filaments are constantly transported toward the perinuclear region while disassembly occurs, giving rise to diffusible subunits for another round of peripheral assembly. Remaining juxtanuclear filaments stabilize and encage the nucleus. Our data suggest that the keratin-filament cycle of assembly and disassembly is a major mechanism of intermediate-filament network plasticity, allowing rapid adaptation to specific requirements, notably in migrating cells. - Introduction The cytoplasmic cytoskeleton of mammalian cells is composed of three major filament networks actin filaments, microtubules and intermediate filaments (IFs). This scaffolding is not simply a static system conferring stability on cells, but is highly dynamic and capable of rapid reorganization in response to various extracellular and intracellular stimuli. Adjustment of actin filaments and microtubules is accomplished through differential regulation of polymerization at either end, depending on the availability of nucleoside-triphosphate-bound soluble subunits and regulatory factors. One prominent mechanism of filament remodelling is treadmilling, which exploits the structural asymmetry of filament ends (Amann and Pollard, 2000; Margolis and Wilson, 1981; Pollard et al., 2000). Thus, subunits dissociate from the minus end and are added to the plus end. IFs are profoundly different from actin filaments and microtubules because they lack polarity as a result of the symmetric composition of their tetrameric subunits. Furthermore, spontaneous self-assembly of tetramers into unit-length filaments (ULFs), followed by compaction and longitudinal annealing, occurs at least in vitro without nucleoside triphosphates and additional cofactors (Herrmann et al., 2007; Kim and Coulombe, 2007). How these in vitro observations relate to the in vivo situation and how assembly is regulated is not understood. IFs are ubiquitous cytoskeletal components that are particularly abundant in epithelial cells. Epithelial IFs are composed of desmosome-anchored keratins, providing a mechanically resilient, complex scaffold (Sivaramakrishnan et al., 2008) that responds quickly to various stimuli through specific structural adaptations. Metabolic, thermal and mechanical stress therefore results in considerable keratin IF (KF) reorganization (Magin et al., 2007; Pekny and Lane, 2007). Recent observations further revealed that the keratin system affects many basic cellular processes, such as growth, proliferation, organelle transport, malignant transformation and stress response, further accentuating the dynamic properties of the keratin network (Magin et al., 2007). Thus, interference with KF dynamics in human disease and transgenic mice leads to reduced resilience of epithelia to mechanical and other challenges with deleterious functional consequences (Arin, 2009; Vijayaraj et al., 2007). For this reason, elucidating the still-unresolved molecular mechanisms of KF-network biogenesis and turnover is a pressing issue. At present, two conceptually different hypotheses are being discussed to explain IF assembly in living cells. The first model, which was recently termed dynamic co-translation, stresses the integration of newly synthesized IF polypeptides at multiple sites throughout the entire network (Chang et al., 2006; Goldman et al., 2008). An alternative model suggests that keratin particles are preferentially generated at distinct loci in the cell periphery, subsequently integrating into the peripheral keratin cytoskeleton (Windoffer et al., 2006; Windoffer et al., 2004). Although both scenarios provide concepts explaining how the network grows, they do not address the problem of how the fully assembled network changes over time. This is crucial for adaptation to varying environmental conditions, for example, during development and under mechanical stress. The goal of our study was to fill the existing gaps in these two hypotheses and to evaluate their contribution to KF-network dynamics. Using live-cell imaging, we discovered a proteinbiosynthesis-independent multistep disassembly and assembly cycle that is continuously active and allows rapid keratin-network adaptation, for instance in migrating cells. Results Keratin-filament network precursor formation occurs preferentially in lamellipodia of migrating cells and persists in the presence of protein biosynthesis inhibitors It was observed by time-lapse fluorescence recording of cell lines producing fluorescent epithelial KFs that KF formation occurs preferentially at free cell edges and adjoining cell borders of stationary cells (Kolsch et al., 2009; Windoffer et al., 2004). After scratch wounding, abundant KF precursors (KFPs) were seen at the leading edge of approaching cells, thereby extending the IF cytoskeleton towards the gap (Fig. 1A; supplementary material Movie 1). This was also noted in single migrating cells (Fig. 1BE; supplementary material Movie 2). KFPs grow by elongation and fusion until integration into the peripheral KF network (for details, see Windoffer et al., 2004; Woll et al., 2005). Considering the abundance of keratin particles in the cell periphery of wounded and migrating cells, we wanted to know whether protein biosynthesis is sufficient to account for keratin assembly, as predicted by the dynamic co-translation model (Chang et al., 2006). Treatment with the translation inhibitors cycloheximide and puromycin, however, did not prevent KFP formation (Fig. 2; supplementary material Movies 3 and 4). KFPs can therefore assemble from a pre-existing keratin pool that is either of significant size or continuously replenished. Keratin filaments translocate continuously toward the nucleus Newly formed KFPs are continuously transported toward the nucleus, relying primarily on intact actin filaments (Kolsch et al., 2009; Woll et al., 2005). To further track KFs after integration into the (...truncated)