The keratin-filament cycle of assembly and disassembly

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.


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;. 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  supplementary material Movie 2). KFPs grow by elongation and fusion until integration into the peripheral KF network (for details, see 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 peripheral network, image-processing algorithms were developed and applied to motility analyses. First, global cellular movement was compensated for. Then, the medians of the translation vectors were computed for each grid point over time, assuming long-term local stability of keratin movement. This revealed coordinated and continuous centripetal keratin movement all the way to the nucleus at an average speed of ~250 nm/minute, with local heterogeneities between 100 and 600 nm/minutes (Fig. 3; supplementary material Movie 5). The motion stopped upon reaching the juxtanuclear region.

Inward-moving keratin filaments disassemble into non-filamentous subunits
Given the movement of the entire keratin system toward the nucleus, one would expect an incremental build up of keratin fluorescence near the nucleus; this is obviously not the case. To test the hypothesis that KFs disassemble, the fluorescence of selected KF subsets was monitored ( Fig. 4; supplementary material Movies 6 and 7). First, regions of interest (ROIs) were defined between prominent peripheral KF bundles. The enclosed KFs translocated continuously towards the nucleus while fusing, generating fewer and thicker bundles (Fig. 4A-F; supplementary material Movie 6). As a result, the ROI size shrank (Fig. 4H). Fluorescence quantification revealed gradual loss that was fast during the first 1-2 hours (~40% loss/hour), affecting the younger peripheral filaments, and slow later (<10% loss/hour), affecting the older thickened filament bundles (Fig. 4I), which eventually stabilize to form the juxtanuclear static cage. This difference is (A-Aٞ) A wound was induced by scratching a confluent monolayer of mammary-epitheliumderived EK18-1 cells that produce fluorescent HK18-YFP with a microinjection needle. Fluorescence images were then recorded until the wound had closed. Selected images are shown in A-Aٞ, demonstrating the continuous and collective convergence of both edges. Multiple KFPs were observed in lamellipodial protrusions of the approaching cells, which can be best appreciated in accompanying supplementary material Movie 1, and are marked by arrows in selected cells. (B-CЉ) Fluorescence images (inverse representation) are shown of a migrating EK18-1 cell with a large lamellipodium containing multiple KFPs that are depicted at higher resolution in C-CЉ. Corresponding phasecontrast images are shown in D-DЉ. The images are taken from a 15 minute time-lapse recording of fluorescence and phase contrast (see supplementary material Movie 2). (E)The kymogram was prepared from supplementary material Movie 2, corresponding to the boxed green area in B. It shows the appearance of multiple KFPs in the lamellipodium in proximity to the plasma membrane (yellow line). The blue line depicts the position of first KFP detection. The subsequent inward-directed movement of KFPs until integration into the peripheral KF network (red line) occurs at similar speed in each instance (slope of line). Scale bars: 10m. best appreciated when the substantial initial fluorescence loss in the blue, green and pink ROIs is compared with the lower initial loss in the red ROI, which was already more central when measurements began (Fig. 4). The apparent fluorescence loss is not caused by bleaching, because overall cellular fluorescence did not decrease. Furthermore, degradation can also be excluded as a major contributing factor, because the fluorescence decrease in entire cells was less than 5% per hour in the presence of puromycin (n4).
To directly monitor disassembly of selected filament bundles, short filament pieces were 'isolated' by bleaching the surrounding areas ( Fig. 4J-N; supplementary material Movie 7). These experiments showed that fluorescence decreased continuously and was not associated with filament fragmentation (i.e. particle formation). We therefore conclude that KFs disintegrate on their way to the nucleus by the release of small, non-filamentous oligomers that cannot be resolved as single particles, but appear as a diffuse weak signal throughout the cytoplasm.

Cells contain a diffusible pool of keratins
To examine the nature of the non-filamentous pool of disassembled KFs, fluorescence recovery after photobleaching (FRAP) analyses were performed. Filament-free regions were selected within bleached areas to measure the mobile fraction of non-filamentous keratins. The mobile fraction was 52.2±12.4% in PK18-5 cells (n5). The diffusion coefficient of this mobile fraction was determined to be 0.88±0.08 m 2 /second for human keratin 18 (HK18)-YFP fusion protein (n9). Furthermore, the diffusion coefficient was not significantly altered in the presence of cytochalasin and nocodazole [1.06±0.31 m 2 /second for HK18-YFP (n8); P0.1052]. In control experiments, the diffusion coefficient for YFP alone was 27.10±7.15 m 2 /second (n20), 2268 Journal of Cell Science 123 (13) which is in close agreement with the values for GFP measured by fluorescence correlation spectroscopy (FCS) (Dross et al., 2009). These findings reveal the existence of a mobile and diffusible cytoplasmic keratin pool whose precise composition and multimerization stage, however, still need to be determined by more sensitive and accurate methods, such as FCS.

Disassembled keratin-filament subunits are reutilized for peripheral keratin-filament assembly
To demonstrate that assembling peripheral KFs are made from material that is derived from disassembled KFs, peripheral cytoplasmic zones of HK18-YFP-producing cells were bleached (Fig. 5A). The mean fluorescence recovery in the peripheral cytoplasm was 72±24% after 15 minutes (n4). During the same time interval, 7.3±2.8% of the fluorescence was lost in the central, non-bleached region, presumably due to translocation of nonfilamentous material to the cell periphery.
Next, an entire cell half was bleached (Fig. 5B). The appearance of fluorescence predominantly in the periphery of the bleached half and its subsequent movement to the cell interior are clearly seen in supplementary material Movie 8. Fluorescence intensities were measured within and outside of the bleached areas, and also in the neighbouring unbleached control cells. As expected, fluorescence increased gradually in the bleached red ROI, with 60% recovery after 1 hour (Fig. 5B). The recovery rate in this ROI is slower than that determined in the previous experiment ( Fig. 5A), because of its more central location and the lower availability of material due to the much more extended bleaching. Remarkably, fluorescence in the unbleached part of the cell decreased during the 60 minute observation period and approached levels similar to those measured in the bleached area, thus providing evidence of continuing KF-network turnover. In comparison, the fluorescence intensity in a corresponding ROI of the adjacent control cell remained almost the same, except for a slight increase that is most probably due to de novo protein biosynthesis (see also Photoactivation experiments were designed to obtain positive proof that filament-bound keratin molecules are released and used for reassembly of new filaments without the need for de novo biosynthesis. After photoactivating human keratin 8 (HK8) hybrids in entire cell halves, new fluorescence appeared in the non-activated parts, the source of which can only be the filament-bound photoactivated molecules from the other cell half (Fig. 5C-D). Within 4 hours, an almost even distribution was noted throughout the cytoplasm (not shown). This is in rough agreement with the bleaching experiments, in which complete exchange of the KF network was observed within 4 hours (supplementary material Movie 8) (data not shown). Furthermore, photoactivated filaments concentrated around the nucleus, whereas inward-moving filaments re-appeared in the cell periphery (see Fig. 5D for quantifications). In support of this, photoactivating only perinuclear filaments resulted in the 'spreading' of fluorescence to the cell periphery ( Fig. 5E-F).

Discussion
Combining the current observations with previous reports on keratin dynamics, key steps of the KF assembly and disassembly cycle have begun to emerge.

Integration
End-on integration of KFPs into the peripheral KF network terminates elongation and thereby introduces branch points into the filament network (Windoffer et al., 2006;Woll et al., 2005).

Bundling
The thickness of the KF bundles increases towards the nucleus because of the lateral association of filament bundles (Figs 3-5; supplementary material Movies 5, 6 and 8). Bundling is regulated by IF-associated polypeptides (Listwan and Rothnagel, 2004) and intrinsic properties of KFs (Lee and Coulombe, 2009).

Vectorial transport
An intact actin-filament system is required for inward-directed KFP transport. The actin retrograde flow and ruffling activity (Small and Resch, 2005) might provide the driving force for centripetal KF movement (Kolsch et al., 2009;Windoffer et al., 2006;Windoffer and Leube, 2001;Woll et al., 2005). Microtubules might also be needed, especially for the subsequent inward motion of the entire network (for a review, see Helfand et al., 2004).

Disassembly
Inward-moving KFs dissolve without the appearance of distinct particles (Fig. 4) either by degradation of KF polypeptides and/or by disassembly into reusable non-filamentous subunits. Although keratin ubiquitylation has been implicated in KF-network turnover (Ku and Omary, 2000) and occurs in stress and disease (Jaitovich et al., 2008;Zatloukal et al., 2007), the second mechanism appears to be the major mode because pre-existing filaments are the source of protein-biosynthesis-independent peripheral filament formation. Of note, the static juxtanuclear filaments are quite stable, as is the case for desmosome-anchored KFs (Strnad et al., 2002).

Diffusion
A rather small though significant mobile fraction of fluorescent keratins with a fast recovery rate was identified in this study. This diffusible keratin pool corresponds, at least in part, to the detergentsoluble pool, which consists primarily of tetramers and also higher order oligomers (Bachant and Klymkowsky, 1996;Chou et al., 1993;Soellner et al., 1985), as indicated by a diffusion coefficient that was significantly lower than that of YFP alone. The identification of diffusing keratins shows that active transport machinery is not needed to deliver disassembled keratins to the were performed in PK18-5 cells producing HK18-YFP. (A)The peripheral network was bleached throughout the entire circumference in a zone extending 5m from the plasma membrane to the cell interior (area between red and yellow lines). The shape of the bleached and non-bleached areas was adjusted by hand to account for cellular shape shifts. Within 15 minutes, considerable fluorescence recovery was observed in the periphery (compare AЈ and AЉ). The histograms below summarize the results of fluorescence quantification in this and three similarly treated cells. The red bars correspond to the average fluorescence recovery in the cell periphery, whereas the yellow bars correspond to the average fluorescence measured in the central cytoplasm. (B)Half of a PK18-5 cell was bleached. Fluorescence recovery measured in the boxed ROI (red), which encompasses mainly central filaments, was ~60% within 60 minutes (red bars in histograms below). By contrast, filaments in the corresponding unbleached ROI (yellow box) decreased within the same time period (yellow bars). For comparison, an ROI in a neighbouring cell that was not bleached at all (blue box) shows slight increase in fluorescence, possibly due to de novo synthesis (blue bars). Corresponding supplementary material Movie 8 presents an image series of 3D reconstructions of the altering fluorescence patterns. (C-F)Photoactivatable GFP fused to keratin 8 (HK8-paGFP) was expressed in PLC cells. HK8-paGFP fluorescence was activated with UV light (405 nm) in different cell regions. Subsequently, fluorescence was monitored at 488 nm by time-lapse fluorescence microscopy. Note the appearance of fluorescence in the non-activated parts of the cells (CЈ,EЈ). In addition, fluorescence concentrated in the perinuclear domain, whereas peripheral fluorescence was generated from new filament formation. Quantification of fluorescence (in percent of total fluorescence) shows gradual depletion of photoactivated regions (yellow) and simultaneous fluorescence increase in non-activated areas (red). D, n3 and F (derived from E,EЈ). N, nucleus. Scale bars: 10m. cell periphery for KF reassembly. This is also supported by the finding that KFP formation persists, although at reduced levels, in the presence of actin filament and microtubule disruptors (Kolsch et al., 2009;Woll et al., 2005). The soluble pool is also available for lateral exchange into existing filaments (Miller et al., 1991), which might explain the slight recovery of bleached filaments in our recordings (e.g. supplementary material Movies 7 and 8).
The revealed keratin cycle is different from dynamic cotranslation (Chang et al., 2006), because it is independent of protein biosynthesis, does not require microtubules and occurs in a spatially defined manner. Our data suggest that the turnover cycle accounts for the vast majority of newly formed filaments. In contrast to the estimated ~25% hourly turnover rate of the keratin cycle, biosynthesis appears to be less than 5% per hour, as inferred from the overall fluorescence increase (e.g. Fig. 5B; Fig. 3N; data not shown). It will be of interest to determine the different contributions of the keratin cycle and dynamic co-translation mechanisms to network plasticity in relation to IF type, cell specialization and specific situation.
The keratin cycle maintains a stationary network at steady state. Preferential assembly at the leading edge of migrating cells leads to an imbalance and subsequent extension of the network towards the lamellipodium. The self-sufficient keratin cycle can be accelerated or slowed by external factors, depending on specific requirements. In support of this, both assembly and disassembly of wild-type and mutant keratins are slowed by p38 mitogen-activated protein kinase inhibitors (Woll et al., 2007). This observation indicates the presence of a basic mechanism whereby signalling affects network reorganization. It exemplifies how keratin-network cycling can be modulated by various types of stress, most notably mechanical stress. Furthermore, regional regulation of the keratin cycle facilitates network restructuring in specific cell topologies. This is needed, for example, in wounded tissues, in which migrating cells not only have to maintain a functional transcellular network but also need to modulate their network architecture during wound closure.

Cell-culture experiments and manipulation
The human hepatocellular-carcinoma-derived epithelial cell line PLC and the stable HK18-YFP-producing subclone PK18-5 were described previously (Strnad et al., 2002). Spontaneously immortalized mouse mammary epithelial cells of line EpH4 and stable transfectants synthesizing HK18-YFP have been presented elsewhere (Kolsch et al., 2009).
Scratch-wounding assays were performed in completely confluent cell monolayers producing homogenously labelled KF networks using a microinjection needle. KFnetwork reorganization during wound closure was monitored by recording timelapse fluorescence images.

Live-cell imaging
Cells were imaged by epifluorescence and confocal laser-scanning microscopy as described . Conversion of paGFP was done by irradiation with five successive scans of 405 nm light at 100% laser intensity.

FRAP experiments
Two types of FRAP experiments were performed to either characterize fast processes concerning non-filamentous keratins or examine the comparatively slow KF dynamics.
To determine the mobile fraction of non-filamentous keratins, a Zeiss 710 confocal laser-scanning microscope was used. Square regions of ~8ϫ15 m were bleached in the peripheral cytoplasm using the 488 nm laser at high intensity. Twenty frames were recorded within the next 10-20 seconds. Within the respective bleached regions, completely filament-free irregularly shaped ROIs of 1-2 m width were chosen and fluorescence recovery in these ROIs was analyzed with the help of Zeiss Zen software.
To determine diffusion coefficients, 5 m diameter ROIs were bleached in the cell periphery and recovery was measured at 0.668 second intervals for 30 seconds. The recovery curves were fitted according to the following equation (Ellenberg et al., 1997): where I (t) is intensity as a function of time, w is the width of ROI (diameter), D is the diffusion constant and t is time. As negative control, cells were fixed with methanol/acetone to demonstrate loss of fluorescence recovery. To exclude active transport, cells were treated with 25 M cytochalasin D and 75 M nocodazole (both from Sigma) 30 minutes prior to bleaching. For statistical evaluation, a two-tailed ttest was performed.
To characterize the slower KF dynamics, a Leica TCS SP5 confocal laser-scanning microscope was employed. Complete fluorescence was measured in single cells and stacks of images containing 4096 grey values (12 bit) were recorded with the 488 nm laser excitation line prior to bleaching.
Step size was between 300 and 500 nm. For bleaching, the 488 nm laser line was applied to different ROIs at high intensity until the fluorescence was below 10% of the pre-bleach level. Starting directly after bleaching, stacks were recorded at regular intervals using the same parameters as for pre-bleach image recording.

Image generation from 3D stacks and quantification of fluorescence
To measure fluorescence intensities in 3D stacks of time series, maximum intensity projections were first generated using ImageProPlus software (Media Cybernetics). The projected images were used for preparing figures and movies. For quantification, ROIs were selected from these projected images. Then, the sum of the grey values was determined in the corresponding ROIs of each image of the z-stack. The values determined for the ROIs in all planes of a stack were then added to calculate the complete 3D fluorescence intensity of a ROI. For comparison of ROI intensities at different time points, it was often necessary to re-define ROIs to account for shifting, size changes and deformation. To correct measurements for background noise, the mean of the grey level per pixel was measured in cell-free regions and subtracted from the grey values determined for pixels measured inside the ROI. Measurements were analyzed with the help of Excel spreadsheets.

Kymograms
To represent movement of KFPs, kymograms were prepared from time-lapse fluorescence recordings. Regions were selected extending from the cell edge to the central cytoplasm. Amira software (Visage Imaging) was used to project the enclosed fluorescence and plot it over time.

Tracking of filaments
Confocal 4D data sets were recorded at 12 bit in cells producing fluorescent keratins. Projections of each time point were used for tracking. ROIs containing a distinct population of filaments were manually defined and Amira software was used to adapt the edges of the chosen ROIs to altered shape in the following frames by visual inspection. From selected time points, the fluorescence intensity of all ROIs was determined by adding their grey values in all 11 planes using ImagePro Plus macros.
In some instances, global cellular movement was compensated by an imageintensity-based registration method (Würflinger et al., 2008) using the normalised sum of squared intensity differences (SSDs). Robustness was increased by embedding the registration in a multiscale Gaussian pyramid framework. Visual assessment of the results revealed no misalignments. Based on the aligned image series, analyses could be restricted to intracellular keratin motion. A z-axis projection of each volume was superimposed by a grid of ROIs at 10-pixel spacing. The overlapping ROIs were of 41-pixel width. Each ROI was rigidly registered to the previous frame by SSD criterion using an exhaustive search for translations of ±20 pixels. SSD was modified by a weighted window function on the ROIs, which dropped linearly from the centre to zero. Thus, the non-rigid character of the data was accounted for and enough structural information was captured by the ROIs, similarly to the work of Wilkie and Vrscay (Wilkie and Vrscay, 2005). Assuming long-term local stability of the keratin flow, the medians of the translation vectors were computed for each grid point over time.
We thank Ursula Wilhelm for expert technical assistance and George Patterson (NIH, Bethesda, USA) for the plasmid encoding paGFP. We also thank Stéphanie Portet (University of Manitoba), Bernd Hoffmann