Keratin Filament Network Research Articles

P38 MAPK-dependent shaping of the keratin cytoskeleton in cultured cells

Plasticity of the resilient keratin intermediate filament cytoskeleton is an important prerequisite for epithelial tissue homeostasis. Here, the contribution of stress-activated p38 MAPK to keratin network organization was examined in cultured cells. It was observed that phosphorylated p38 colocalized with keratin granules that were rapidly formed in response to orthovanadate. The same p38p recruitment was noted during mitosis, in various stress situations and in cells producing mutant keratins. In all these situations keratin 8 became phosphorylated on S73, a well-known p38 target site. To demonstrate that p38-dependent keratin phosphorylation determines keratin organization, p38 activity was pharmacologically and genetically modulated: up-regulation induced keratin granule formation, whereas down-regulation prevented keratin filament network disassembly. Furthermore, transient p38 inhibition also inhibited keratin filament precursor formation and mutant keratin granule dissolution. Collectively, the rapid and reversible effects of p38 activity on keratin phosphorylation and organization in diverse physiological, stress, and pathological situations identify p38-dependent signalling as a major intermediate filament–regulating pathway.

Keratin 20 Serine 13 Phosphorylation Is a Stress and Intestinal Goblet Cell Marker

Keratin polypeptide 20 (K20) is an intermediate filament protein with preferential expression in epithelia of the stomach, intestine, uterus, and bladder and in Merkel cells of the skin. K20 expression is used as a marker to distinguish metastatic tumor origin, but nothing is known regarding its regulation and function. We studied K20 phosphorylation as a first step toward understanding its physiologic role. K20 phosphorylation occurs preferentially on serine, with a high stoichiometry as compared with keratin polypeptides 18 and 19. Mass spectrometry analysis predicted that either K20 Ser(13) or Ser(14) was a likely phosphorylation site, and Ser(13) was confirmed as the phospho-moiety using mutation and transfection analysis and generation of an anti-K20-phospho-Ser(13) antibody. K20 Ser(13) phosphorylation increases after protein kinase C activation, and Ser(13)-to-Ala mutation interferes with keratin filament reorganization in transfected cells. In physiological contexts, K20 degradation and associated Ser(13) hyperphosphorylation occur during apoptosis, and chemically induced mouse colitis also promotes Ser(13) phosphorylation. Among mouse small intestinal enterocytes, K20 Ser(13) is preferentially phosphorylated in goblet cells and undergoes dramatic hyperphosphorylation after starvation and mucin secretion. Therefore, K20 Ser(13) is a highly dynamic protein kinase C-related phosphorylation site that is induced during apoptosis and tissue injury. K20 Ser(13) phosphorylation also serves as a unique marker of small intestinal goblet cells.

Focal adhesions are hotspots for keratin filament precursor formation

Recent studies showed that keratin filament (KF) formation originates primarily from sites close to the actin-rich cell cortex. To further characterize these sites, we performed multicolor fluorescence imaging of living cells and found drastically increased KF assembly in regions of elevated actin turnover, i.e., in lamellipodia. Abundant KF precursors (KFPs) appeared within these areas at the distal tips of actin stress fibers, moving alongside the stress fibers until their integration into the peripheral KF network. The earliest KFPs were detected next to actin-anchoring focal adhesions (FAs) and were only seen after the establishment of FAs in emerging lamellipodia. Tight spatiotemporal coupling of FAs and KFP formation were not restricted to epithelial cells, but also occurred in nonepithelial cells and cells producing mutant keratins. Finally, interference with FA formation by talin short hairpin RNA led to KFP depletion. Collectively, our results support a major regulatory function of FAs for KF assembly, thereby providing the basis for coordinated shaping of the entire cytoskeleton during cell relocation and rearrangement.

Keratin 18 overexpression but not phosphorylation or filament organization blocks mouse Mallory body formation

Several human liver diseases are associated with formation of Mallory body (MB) inclusions. These hepatocyte cytoplasmic deposits are composed primarily of hyperphosphorylated keratins 8 and 18 (K8/K18). Feeding a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet is a well-established mouse model of MBs. K8 overexpression, and K8-null or K18-null mouse models, indicate that a K8-greater-than-K18 expression ratio is critical for MB formation. We used established transgenic mouse models to study the effect of K18 overexpression and phosphorylation, or keratin filament disorganization, on MB formation. Five mouse lines were used: nontransgenic, those that overexpress wild-type K18 or the K18 phosphorylation mutants Ser33-to-Ala (S33A) or Ser52-to-Ala (S52A), and mice that overexpress K18 Arg89-to-Cys, which causes collapse of the keratin filament network into dots. DDC feeding induced MBs in nontransgenic livers, but MBs were rarely seen in any of the K18 transgenic mice. Wild-type K18 overexpression protected mice from DDC-induced liver injury. K18 overexpression protects mice from MB formation and from DDC-induced liver injury, which supports the importance of the K8-to-K18 ratio in MB formation. The effect of K18 on MB formation is independent of hepatocyte keratin filament organization or K18 Ser33/Ser52 phosphorylation. Keratin filament collapse, which is a major risk for acute liver injury, is well tolerated in the context of chronic DDC-mediated liver injury.

Fitting of random tessellation models to keratin filament networks

The role of specific structural patterns in keratin filament networks for regulating biophysical properties of epithelial cells is poorly understood. This is at least partially due to a lack of methods for the analysis of filament network morphology. We have previously developed a statistical approach to the analysis of keratin filament networks imaged by scanning electron microscopy. The segmentation of images in this study resulted in graph structures, i.e. tessellations, whose structural characteristics are now further investigated by iteratively fitting geometrical statistical models. An optimal model as well as corresponding optimal parameters are detected from a given set of possible random tessellation models, i.e. Poisson–Line tessellations (PLT), Poisson–Voronoi tessellations (PVT) and Poisson–Delaunay tessellations (PDT). Using this method, we investigated the remodeling of keratin filament networks in pancreatic cancer cells in response to transforming growth factor α (TGF α ), which is involved in pancreatic cancer progression. The results indicate that the fitting of random tessellation models represents a suitable method for the description of complex filament networks.

Quantitative analysis of keratin filament networks in scanning electron microscopy images of cancer cells

The keratin filament network is an important part of the cytoskeleton. It is involved in the regulation of shape and viscoelasticity of epithelial cells. The morphology of keratin networks depends on post-translational modifications of keratin monomers. In-vitro studies indicated that network characteristics, such as filament crosslink density, determines the biophysical properties of the filament network. This report presents a quantitative method for the morphological analysis of keratin filament networks. Visualization of filaments was based on prefixation extraction of epithelial cells and scanning electron microscopy (SEM). SEM images were processed by a skeletonization algorithm to obtain a graph structure that represents individual filaments as well as their connections. This method was applied to investigate the effects of transforming growth factor alpha (TGFalpha) on the morphology of keratin networks in pancreatic cancer cells. TGFalpha contributes to pancreatic cancer progression and activates signalling pathways phosphorylating keratin monomers. Using this new method, a significant alteration to the keratin network morphology could be detected in response to TGFalpha.

Striate palmoplantar keratoderma arising from desmoplakin and desmoglein 1 mutations is associated with contrasting perturbations of desmosomes and the keratin filament network.

Several hereditary human diseases are now known to be caused by distinct mutations in genes encoding various desmosome components. Although the effects of some of these mutant genes have been analysed by targeted disruption experiments in mouse models, little is known about the cell and tissue changes in affected human patients. To investigate the effects of heterozygous nonsense mutations in desmoplakin (Dp) and desmoglein (Dsg) 1 which cause the autosomal dominant disorder striate palmoplantar keratoderma (SPPK), focusing on changes in desmosome structure and composition and the associated keratin intermediate filament (KIF) network in palm skin, and in cultured keratinocytes generated from the same site. We analysed palm and nonpalm skin sections from four SPPK patients with Dp mutations and one patient with a Dsg1 mutation with respect to tissue and subcellular morphologies, and correlated the in vivo and in vitro findings. Using electron microscopy, we found abnormalities of desmosomes and cell-cell adhesion in the suprabasal layers in the epidermis from patients with both Dsg1- and Dp-associated SPPK. These changes were more advanced in skin from patients with Dp mutations. Both Dp and Dsg1 mutations were accompanied by significantly reduced numbers of desmosomes in the suprabasal layers, while decreased desmosome size was evident only in Dsg1-associated SPPK. Confocal microscopy analysis showed marked differences in the expression of keratins and of desmosome components, both between the two types of SPPK, and between SPPK and normal skin. The expression of keratins K5, K14 and K10 was reduced in Dsg1-associated SPPK skin, whereas perinuclear aggregation of keratin filaments was more evident in Dp-associated SPPK. In both types of SPPK upregulation of K16 was pronounced and involucrin labelling was abnormal. Mutations in Dp and Dsg1 genes causing SPPK may be associated with perturbations in epidermal differentiation accompanied by a marked disruption of several components of the epidermal scaffold including desmosomes and the KIF network.

Human keratin 8 mutations that disturb filament assembly observed in inflammatory bowel disease patients

We have identified miss-sense mutations in keratin 8 in a subset of patients with inflammatory bowel disease (Crohn disease and ulcerative colitis). Inflammatory bowel diseases are a group of disorders that are polygenic in origin and involve intestinal epithelial breakdown. We investigated the possibility that these keratin mutations might contribute to the course of the disease by adversely affecting the keratin filament network that provides mechanical support to cells in epithelia. The mutations (Gly62 to Cys, Ile63 to Val and Lys464 to Asn) all lie outside the major mutation hotspots associated with severe disease in epidermal keratins, but using a combination of in vitro and cell culture assays we show that they all have detrimental effects on K8/K18 filament assembly in vitro and in cultured cells. The G62C mutation also gives rise to homodimer formation on oxidative stress to cultured intestinal epithelial cells, and homodimers are known to be polymerization incompetent. Impaired keratin assembly resulting from the K8 mutations found in some inflammatory bowel disease patients would be predicted to affect the maintenance and re-establishment of mechanical resilience in vivo, as required during keratin cytoskeleton remodeling in cell division and differentiation, which may lead to epithelial fragility in the gut. Simple epithelial keratins may thus be considered as candidates for genes contributing to a risk of inflammatory bowel disease.

Identification of novel principles of keratin filament network turnover in living cells.

It is generally assumed that turnover of the keratin filament system occurs by exchange of subunits along its entire length throughout the cytoplasm. We now present evidence that a circumscribed submembranous compartment is actually the main site for network replenishment. This conclusion is based on the following observations in living cells synthesizing fluorescent keratin polypeptides: 1) Small keratin granules originate in close proximity to the plasma membrane and move toward the cell center in a continuous motion while elongating into flexible rod-like fragments that fuse with each other and integrate into the peripheral KF network. 2) Recurrence of fluorescence after photobleaching is first seen in the cell periphery where keratin filaments are born that translocate subsequently as part of the network toward the cell center. 3) Partial keratin network reformation after orthovanadate-induced disruption is restricted to a distinct peripheral zone in which either keratin granules or keratin filaments are transiently formed. These findings extend earlier investigations of mitotic cells in which de novo keratin network formation was shown to originate from the cell cortex. Taken together, our results demonstrate that the keratin filament system is not homogeneous but is organized into temporally and spatially distinct subdomains. Furthermore, the cortical localization of the regulatory cues for keratin filament turnover provides an ideal way to adjust the epithelial cytoskeleton to dynamic cellular processes.

Keratin 8 and 18 hyperphosphorylation is a marker of progression of human liver disease.

Keratin 8 and 18 (K8/18) phosphorylation plays a significant and site-specific role in regulating keratin filament organization, association with binding proteins, and modulation of cell cycle progression. Keratin hyperphosphorylation correlates with exposure to a variety of stresses in cultured cells and in mouse models of liver, pancreatic, and gallbladder injury, and it is found in association with mouse and human Mallory bodies. We asked whether K8/18 phosphorylation correlates with human liver disease progression by analyzing liver explants and biopsies of patients with chronic noncirrhotic hepatitis C virus (HCV) or cirrhosis. We also examined the effect of HCV therapy with interleukin-10 on keratin phosphorylation. Using site-specific antiphosphokeratin antibodies we found keratin hyperphosphorylation on most K8/18 sites in all cirrhotic liver explants tested and in most liver biopsies from patients with chronic HCV infection. Immunofluorescence staining of precirrhotic HCV livers showed focal keratin hyperphosphorylation and limited reorganization of keratin filament networks. In cirrhotic livers, keratin hyperphosphorylation occurred preferentially in hepatic nodule cells adjacent to bridging fibrosis and associated with increased stress kinase activation and apoptosis. Histological and serological improvement after interleukin-10 therapy was accompanied by normalization of keratin hyperphosphorylation on some sites in 7 of 10 patients. In conclusion, site-specific keratin phosphorylation in liver disease is a progression marker when increased and a likely regression marker when decreased.

HSP25 is involved in two steps of the differentiation of PAM212 keratinocytes.

HSP25 is a member of the small heat shock protein family. This 25-kDa protein exhibits a highly specific distribution during mouse embryonic development. Although multiple functions have been proposed for HSP25, the role it plays during differentiation is still unknown. High levels of HSP25 can be detected in embryonic and adult skin. During epidermis differentiation, the concentration of HSP25 increases with the distance of keratinocytes from the basal layer, in parallel with the extent of keratinization. We used an ex vivo cellular system, PAM212 cells, to analyze quantitatively and qualitatively the dynamics of HSP25 production and phosphorylation during the differentiation of keratinocytes. Our observations suggest that HSP25 is involved in two steps of PAM212 keratinocyte differentiation. Shortly after the induction of differentiation, a transient hyperphosphorylation of HSP25 seems to be essential for the expression of differentiation markers. Later, the chaperone-active form of HSP25 is organized progressively into characteristic aggregates involved in the dynamics of keratin filament networks.

Trichohyalin Mechanically Strengthens the Hair Follicle: MULTIPLE CROSS-BRIDGING ROLES IN THE INNER ROOT SHEATH

Trichohyalin is expressed in specialized epithelia that are unusually mechanically strong, such as the inner root sheath cells of the hair follicle. We have previously shown that trichohyalin is sequentially subjected to post-synthetic modifications by peptidylarginine deaminases, which convert many of its arginines to citrullines, and by transglutaminases, which introduce intra- and interprotein chain cross-links. Here we have characterized in detail the proteins to which it becomes cross-linked in vivo in the inner root sheath of the mouse hair follicle. We suggest that it has three principal roles. First, it serves as an interfilamentous matrix protein by becoming cross-linked both to itself and to the head and tail end domains of the inner root sheath keratin intermediate filament chains. A new antibody reveals that arginines of the tail domains of the keratins are modified to citrullines before cross-linking, which clarifies previous studies. Second, trichohyalin serves as a cross-bridging reinforcement protein of the cornified cell envelope of the inner root sheath cells by becoming cross-linked to several known or novel barrier proteins, including involucrin, small proline-rich proteins, repetin, and epiplakin. Third, it coordinates linkage between the keratin filaments and cell envelope to form a seamless continuum. Together, our new data document that trichohyalin is a multi-functional cross-bridging protein that functions in the inner root sheath and perhaps in other specialized epithelial tissues by conferring to and coordinating mechanical strength between their peripheral cell envelope barrier structures and their cytoplasmic keratin filament networks.

Disruption of the suprabasal keratin network by mutation M150T in the helix initiation motif of keratin 10 does not affect cornified cell envelope formation in human epidermis.

Keratin 10 (K10) is known to be tightly bound to the cornified cell envelope (CCE) and this binding is thought to play an important role in enhancing the structural integrity of the cornified cells. Bullous congenital ichthyosiform erythroderma (BCIE) is a genetic disorder of keratinization caused by gene mutations in the conserved sequences of keratin 1 (K1) or K10, which leads to abnormal suprabasal keratin network assembly. In BCIE patients' skin, the keratin network abnormalities make the upper spinous and granular keratinocytes fragile and result in blister formation. However, the exact pathomechanism of the hyperkeratosis seen in BCIE is still unknown. The involvement of the CCE in the pathomechanism of hyperkeratosis in BCIE is controversial. Abnormal CCE assembly may cause hyperkeratosis as reported in cases of lamellar ichthyosis. Binding of K10 to CCE is thought to be a vital connection between the suprabasal keratin filament network and CCE. We hypothesize that abnormal suprabasal keratin assembly caused by either K1 or K10 mutations can disrupt CCE formation, resulting in the hyperkeratosis observed in BCIE. To clarify whether K10 and keratin network defects affect CCE formation in vivo, the ultrastructural and immunohistological features of CCE were studied in the epidermis of two Japanese BCIE patients from two independent families carrying an identical missense mutation M150T in the helix initiation motif of K10. Ultrastructurally, a 15-nm-thick, dense, normal-appearing CCE was formed at the cell periphery of the keratinized epidermal cells. Light and electron microscopic immunolabeling revealed that the major CCE precursor proteins, involucrin and loricrin, were normally distributed and restricted to CCE of the epidermis. Immunofluorescent labeling showed that epidermal TGases, TGase 1, TGase 2 and TGase 3, were expressed normally in the epidermis. These findings suggest that a normal CCE is formed during the process of human epidermal keratinization, even if the suprabasal keratin filament network is disrupted as with this particular K10 mutation, M150T in BCIE.

Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene. Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3' end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN). No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms. Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells.

Light-induced Resistance of the Keratin Network to the Filament-disrupting Tyrosine Phosphatase Inhibitor Orthovanadate

Epidermal keratinocytes respond to low-dose light irradiation by inducing signaling cascades that lead to long-term effects on gene transcription thereby protecting cells against damage. In contrast, little is known about immediate light-induced alterations of structural proteins. We have made the intriguing observation that light produces fundamental changes in the properties of the keratin filament system of cultured epidermoid A-431 cells. A short light exposure (1-10 min) causes the keratin cytoskeleton to become immediately resistant to the tyrosine phosphatase inhibitor orthovanadate, which otherwise disrupts the keratin filament network completely in just a few minutes. This protective effect is inducible throughout the entire visible spectrum and is elicited by normal room light (<200 Lux). Exposure of cells to monochromatic light of various wavelengths is therefore equally effective. In addition, the acquisition of orthovanadate resistance has been directly monitored in living cells; a partially disrupted keratin cytoskeleton recovers to a completely filamentous network in half an hour. Finally, the protective light effect is largely reversed in 2 h and can be mimicked by preincubation with the p38 kinase inhibitor SB203580. In contrast, the mitogen-activated protein kinase inhibitor PD98059 and epidermal growth factor inhibit orthovanadate action to a lesser extent. Taken together, these observations suggest a stabilizing function of light on the keratin filament network; this may be of relevance to the treatment of skin diseases with reduced keratin stability.

The human integrin β4B and β4C variants are not expressed in a tissue-specific manner

The human integrin β4B and β4C variants are not expressed in a tissue-specific manner

Compound Heterozygosity for Non-Sense and Mis-Sense Mutations in Desmoplakin Underlies Skin Fragility/Woolly Hair Syndrome

The constitutive desmosomal plaque protein desmoplakin plays a vital part in keratinocyte adhesion in linking the transmembranous desmosomal cadherins to the cytoplasmic keratin filament network. Recently, mutations in desmoplakin have been shown to underlie some cases of the autosomal dominant disorder, striate palmoplantar keratoderma, as well as an autosomal recessive condition characterized by dilated cardiomyopathy, woolly hair, and keratoderma. Here, we describe two unrelated individuals with a new autosomal recessive genodermatosis characterized by focal and diffuse palmoplantar keratoderma, hyperkeratotic plaques on the trunk and limbs, varying degrees of alopecia, but no apparent cardiac anomalies. Mutation screening of desmoplakin demonstrated compound heterozygosity for a non-sense/mis-sense combination of mutations in both cases, C809X/N287K and Q664X/R2366C, respectively. Heterozygous carriers of any of these mutations displayed no phenotypic abnormalities. Immunohistochemistry of skin biopsies from both affected individuals revealed that desmoplakin was not just located at the cell periphery but there was also cytoplasmic staining. In addition, electron microscopy demonstrated acantholysis throughout all layers of the skin, focal detachment of desmosomes into the intercellular spaces, and perinuclear condensation of the suprabasal keratin intermediate filament network. Clinicopathologic and mutational analyses therefore demonstrate that desmoplakin haploinsufficiency can be tolerated in some cases, but that in combination with a mis-sense mutation on the other allele, the consequences are a severe genodermatosis with specific clinical manifestations.

A 'hot-spot' mutation alters the mechanical properties of keratin filament networks.

Keratins 5 and 14 polymerize to form the intermediate filament network in the progenitor basal cells of many stratified epithelia including epidermis, where it provides crucial mechanical support. Inherited mutations in K5 or K14 result in epidermolysis bullosa simplex (EBS), a skin-fragility disorder. The impact that such mutations exert on the intrinsic mechanical properties of K5/K14 filaments is unknown. Here we show, by using differential interference contrast microscopy, that a 'hot-spot' mutation in K14 greatly reduces the ability of reconstituted mutant filaments to bundle under crosslinking conditions. Rheological assays measure similar small-deformation mechanical responses for crosslinked solutions of wild-type and mutant keratins. The mutation, however, markedly reduces the resilience of crosslinked networks against large deformations. Single-particle tracking, which probes the local organization of filament networks, shows that the mutant polymer exhibits highly heterogeneous structures compared to those of wild-type filaments. Our results indicate that the fragility of epithelial cells expressing mutant keratin may result from an impaired ability of keratin polymers to be crosslinked into a functional network.

A Novel Asparagine→Aspartic Acid Mutation in the Rod 1A Domain in Keratin 2e in a Japanese Family with Ichthyosis Bullosa of Siemens

Ichthyosis bullosa of Siemens is a unique type of congenital ichthyosis characterized by mild hyperkeratosis over the flexural areas and blister formation after mechanical trauma and superficial denuded areas in the hyperkeratotic skin. Recently, mutations in the helix initiation or termination motifs of keratin 2e (KRT2E) have been described in ichthyosis bullosa of Siemens patients. The majority of the mutations reported to date lie in the 2B region. We report a novel amino acid substitution mutation (asparagine-->aspartic acid) in codon 192 at the conserved 1A helix initiation site of the rod domain of KRT2E in a Japanese family with ichthyosis bullosa of Siemens. Our data indicate aspartic acid substitution in codon 192 in the 1A helix initiation site is deleterious to keratin filament network integrity and leads to ichthyosis bullosa of Siemens phenotype.

Plakophilin, armadillo repeats, and nuclear localization.

Plakophilins are armadillo-repeat containing proteins, identified through their localization to desmosomes. Expressed in a wide range of tissues, plakophilins are largely nuclear in most cell types [Schmidt et al. (1997) Cell Tissue Res 290:481; Mertens et al. (1996) J. Cell Biol 135:1009]. Using Xenopus embryos and cultured A6 cells, together with myc- and green fluorescent protein (GFP)-tags, we found that both the N-terminal, non-armadillo repeat "head" and the C-terminal armadillo repeat-containing regions can enter nuclei. The "arm" repeat domain is predominantly cytoplasmic and concentrated at the cell cortex, whereas the head and full-length polypeptides are concentrated in the nucleus. The head domain can also be seen to decorate and disrupt keratin filament network organization in some cells. In the course of these studies, we found that the distribution of the myc-epitope and green fluorescence differed in fixed cells, e.g., while the green fluorescence of a myc- and GFP-tagged head domain polypeptide was usually exclusively nuclear, a substantial fraction of the myc-immunoreactivity was cytoplasmic. Treating cells with the translation inhibitor cycloheximide reduces the cytoplasmic myc-signal, suggesting that it represented nascent polypeptides awaiting folding and nuclear import. Based on these types of experiments, GFP can be seen as a marker of the distribution of the mature form of the tagged polypeptide.