Dissection of keratin dynamics: different contributions of the actin and microtubule systems

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Abstract

It has only recently been recognized that intermediate filaments (IFs) and their assembly intermediates are highly motile cytoskeletal components with cell-type- and isotype-specific characteristics. To elucidate the cell-type-independent contribution of actin filaments and microtubules to these motile properties, fluorescent epithelial IF keratin polypeptides were introduced into non-epithelial, adrenal cortex-derived SW13 cells. Time-lapse fluorescence microscopy of stably transfected SW13 cell lines synthesizing fluorescent human keratin 8 and 18 chimeras HK8-CFP and HK18-YFP revealed extended filament networks that are entirely composed of transgene products and exhibit the same dynamic features as keratin systems in epithelial cells. Detailed analyses identified two distinct types of keratin motility: (I) Slow (∼0.23 μm/min), inward-directed, continuous transport of keratin filament precursor particles from the plasma membrane towards the cell interior, which is most pronounced in lamellipodia. (II) Fast (∼17 μm/min), bidirectional and intermittent transport of keratin particles in axonal-type cell processes. Disruption of actin filaments inhibited type I motility while type II motility remained. Conversely, microtubule disruption inhibited transport mode II while mode I continued. Combining the two treatments resulted in a complete block of keratin motility. We therefore conclude that keratin motility relies both on intact actin filaments and microtubules and is not dependent on epithelium-specific cellular factors.

Introduction

The framework of the cytoplasmic cytoskeleton is composed of three major filament systems. They include the 2–5-nm actin-based microfilaments, the 25-nm microtubules that are composed of tubulin heterodimers, and the compositionally most diverse intermediate filaments (IFs) of 8–12 nm diameter. When considering the various functions that rely on the cytoskeleton, e.g., migration, cell division, vesicle trafficking, or stress response, it appears imperative that the reactions of the cytoskeletal components are co-ordinated and that no part acts entirely on its own. In accordance, it was reported a long time ago that drugs that specifically disrupt either actin filaments or microtubules also affect the organization of the IF system (e.g., Celis et al., 1984; Goldman, 1971; Hynes and Destree, 1978; Osborn et al., 1980). Yet, other authors found that only microtubule but not actin disruption leads to IF reorganization (Gordon et al., 1978). To complicate the situation further, several groups did not detect any alterations in IF distribution in certain situations when either of the other cytoskeletal filament systems was destroyed and thus used this very property to define specific IF types (e.g., Knapp et al., 1983a, Knapp et al., 1983b; Osborn et al., 1977; Sun and Green, 1978) although the combined destruction of actin filaments and microtubules was accompanied by significant IF alterations (Knapp et al., 1983a, Knapp et al., 1983b). As a result, the variably occurring structural IF alterations, which  in contrast to the other cytoskeletal filament systems  did not lead to disassembly but rather to reorganization and formation of perinuclear filament bundles, may be attributed to specific properties of individual IF types in different cellular environments or could be interpreted as non-specific effects.

Recent years have provided overwhelming evidence that molecular ties exist between all cytoskeletal filaments. Static links are provided by the plakins, a complex family of large molecules that facilitate physical contact between different filament types, cell adhesion sites and cytoplasmic organelles (Fuchs and Karakesisoglou, 2001; Leung et al., 2002). Probably the most prominent plakin is plectin, which acts as a general cytoskeletal cross-linker with multiple interaction types depending on the splice variants present in a given cell (Steinbock and Wiche, 1999; Wiche, 1998). In addition, dynamic attachments are established by motor proteins that enable IFs and their precursors to move in relation to the other cytoskeletal filaments (review in Helfand et al., 2003a).

Imaging of IFs in living cells has revealed that they are highly dynamic cell components both in terms of motility and turnover (recent reviews in Helfand, 2003a, Helfand, 2004). This also includes the epithelial keratin IFs, which are essential stabilizers of the epithelial cytoskeleton by providing resilience to mechanical stress (Coulombe and Wong, 2004; Herrmann et al., 2003). Various types of motility have been described for keratin filaments (KFs) and keratin particles by several groups (Liovic et al., 2003; Werner et al., 2004; Windoffer et al., 2002, Windoffer et al., 2004; Windoffer and Leube, 1999, Windoffer and Leube, 2001, Windoffer and Leube, 2004; Yoon et al., 2001), and it has been shown that the dynamic keratin organization is modulated by cellular phosphorylation (Strnad et al., 2001, Strnad et al., 2002, Strnad et al., 2003).

To examine the importance of actin filaments and microtubules for the motile properties of keratin assemblies, we monitored the effects of actin filament and microtubule disruptions in living vulva carcinoma-derived A-431 cells producing fluorescent keratin 13 chimeras (Windoffer and Leube, 1999). We observed that adding of cytochalasin D resulted in network retraction leading to partial collapse of the extended KF system. On the other hand, treatment of the same cell line with nocodazole led to a marked decrease in KF network oscillations (see also Liovic et al., 2003; Yoon et al., 2001). Furthermore, we examined the effects of microtubule- and actin-disrupting drugs on the de novo formation of the KF cytoskeleton at the end of mitosis (Windoffer and Leube, 2001). Coincidentally, KFs are completely disassembled into soluble subunits and granules at the beginning of mitosis in A-431 cells. This material is subsequently used for the rebuilding of a new KF network. Adding both cytochalasin D and nocodazole prevented formation of a typical KF network. In the presence of cytochalasin, elongated clumped material was formed that moved toward the cell center and lacked anchorage to the cell cortex. On the other hand, incubation with nocodazole resulted in restricted fluorescence enrichment in the cell periphery, and it seemed that granules dissolved into diffuse, possibly filamentous structures. Taken together, these results suggested that both actin filaments and microtubules are important for dynamic KF organization, yet in a different way that may not always be apparent in still pictures.

A dynamic interaction between microtubules and IFs has also been recognized for neurofilaments, peripherin and vimentin (Chan et al., 2003; Helfand, 2002, Helfand, 2003b; Martys et al., 1999; Prahlad et al., 1998, Prahlad et al., 2000; Shah et al., 2000; Yabe et al., 1999, Yabe et al., 2000, Yabe et al., 2001; Yoon et al., 2001). It has been pointed out, however, especially in direct comparisons between keratin and vimentin IFs, that significant differences exist with respect to dynamic properties and transport specificities (Yoon et al., 2001).

To further define principal aspects of the interrelationship of the IF cytoskeleton with microtubules and actin filaments in a cell-type-independent context with improved technology, we decided to study the behavior of keratin IF proteins in SW13 cells, that usually lack IF proteins altogether (Hedberg and Chen, 1986). By adding drugs that selectively inactivate actin filaments or microtubules, the contribution of each system to the dynamic behavior of KFs and KF precursors was determined in SW13 cells that were stably transfected with cDNAs coding for chimeric keratin-fluorescent protein pairs.

Section snippets

cDNA cloning

For construction of HK8ΔT-EGFP, a ∼1.3-kb EcoRI/SacI fragment was excised from HCK8-containing human keratin 8 cDNA in vector pDs5 (Hofmann and Franke, 1997). The EcoRI site was blunted and the fragment subcloned into SmaI/SacI-cleaved vector pSP64 (Promega, Madison, WI), thereby generating plasmid pHK8ΔT. The ∼1.3-kb keratin 8 cDNA fragment was excised from this plasmid with BamHI/EcoRI and inserted into BglII/EcoRI-cleaved vector pEGFP-N3 (Clontech, Palo Alto, CA). The encoded polypeptide

In vivo assembly of keratin-fluorescent protein hybrids in non-epithelial SW13 cells into extended keratin filament networks

We have previously observed that transfected fluorescent keratin polypeptides form characteristic cytoplasmic networks in non-epithelial, adrenal cortex-derived SW13 cells (Windoffer et al., 2004; see also Yoon et al., 2001) which are devoid of any cytoplasmic IF (Hedberg and Chen, 1986). To further study properties and characteristics of these ectopic networks that are entirely composed of hybrid polypeptides, we established clonal cell lines producing keratin 8 and 18 chimeras HK8-CFP and

Discussion

By using live cell imaging, we readdressed a long-standing debate regarding the manner in which the actin and microtubule networks affect the organization of the keratin system. We obtained strong evidence that both are intricately interlaced with the dynamics of the keratin network and showed that each contributes to keratin motility in a different and highly specific way in a cell-type-independent fashion.

Acknowledgements

The expert technical assistance of Ursula Wilhelm is gratefully acknowledged. The work was supported in part by the German Research Council. The senior author wishes to express his gratitude to Professor Franke for instilling his enthusiasm for the endless and miraculous beauty of nature which provides many exciting answers and even more fascinating enigma.

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