Keratin polypeptides 8 and 18 (K8/18) are the major intermediate filament proteins of simple-type epithelia. K18 Ser-33 phosphorylation regulates its binding to 14-3-3 proteins during mitosis. We studied the significance of keratin binding to 14-3-3 in transgenic mice that overexpress wild-type or Ser-33->>Ala (S33A) K18. In S33A but not wild-type K18-overexpressing mice, pancreatic acinar cell keratin filaments retracted from the basal nuclear region and became apically concentrated. In contrast, K18 S33A had a minimal effect on hepatocyte keratin filament organization. Partial hepatectomy of K18-S33A-overexpressing mice did not affect liver regeneration but caused limited mitotic arrest, accumulation of abnormal mitotic figures, dramatic fragmentation of hepatocyte keratin filaments, with retention of a speckled 14-3-3zeta mitotic cell nuclear-staining pattern that usually becomes diffuse during mitosis. Hence, K18 Ser-33 phosphorylation regulates keratin filament organization in simple-type epithelia in vivo. Keratin binding to 14-3-3 may partially modulate hepatocyte mitotic progression, in association with nuclear redistribution of 14-3-3 proteins during mitosis.

A cDNA clone of a keratin-related, intermediate filament protein, designated Endo B, was constructed from size-fractionated parietal endodermal mRNA and characterized. The 1466-nucleotide cDNA insert contains an open reading frame of 1272 nucleotides that would result in 5' and 3' noncoding sequences of 54 and 60 nucleotides, respectively. The predicted amino acid composition, molecular weight (47,400), and peptide pattern correlate well with data obtained on the isolated protein. The predicted amino acid sequence fits easily into the general domain structure suggested for all intermediate filament proteins with a unique amino-terminal head domain, a large conserved central domain of predominantly alpha-helical structure, and a relatively unique carboxyl-terminal or tail domain. Over the entire molecule, Endo B is 43% identical with human 52-kDa epidermal type I keratin. However, over two of the three regions contained in the central domain that are predicted to form coiled-coil structures, the Endo B is 54-68% identical with other type I keratin sequences. This homology, along with the presence of the completely conserved sequence DNARLAADDFR-KYE, which is found in all type I keratins, permits the unambiguous identification of Endo B as a type I keratin. Comparison of the Endo B sequence to other intermediate filament proteins reveals 22 residues which are identical in all intermediate filament proteins regardless of whether filament formation requires only one type of protein subunit (vimentin, desmin, glial fibrillar acidic protein, or a neurofilament protein) or two dissimilar types (type I and type II keratins). Endo B mRNA was detectable in RNA isolated from F9 cells treated with retinoic acid for 48 h. Approximately three to five genes homologous to Endo B were detected in the mouse genome.

OBJECTIVE

To improve our understanding of how retinal pigment epithelial (RPE) cells behave in vivo and to establish similarities with dedifferentiation and adaptive events observed in RPE cells cultured under simulated intraocular pathologic conditions. At the same time, to examine the origin of epithelioid-shaped and fibroblast/fusiform-shaped cells in epiretinal membranes (ERM) from proliferative vitreoretinopathy (PVR).

METHODS

Cells of ERM were studied by electron-immunocytochemical techniques, using simple, double, and triple immunostaining for cytokeratins (CK), vimentin (Vim), and glial fibrillary acidic protein (GFAP). Ultrastructural morphology analysis was also carried out. Adult human RPE cells were obtained and cultured with normal and pathologic vitreous from proliferative vitreoretinal disorders, subretinal fluid aspirates from retinal detachment, and normal human serum. Their cytoskeleton was fractionated at 7 (early cultures) and 24 (late cultures) days of culture, electrophoresed, immunoblotted for intermediate filament proteins, and quantified by densitometric analysis for each condition. Changes in phenotype characteristics were also evaluated.

RESULTS

Epithelioid-shaped and fibroblast/fusiform-shaped cells, resembling RPE cells, expressed CK-Vim-GFAP simultaneously as intermediate filament proteins in their cytoskeleton. RPE cells in culture also expressed CK-Vim-GFAP and changed from an epithelial shape to a migratory fibroblast/fusiform-shaped phenotype in the presence of subretinal fluid aspirates and pathologic vitreous from proliferative intraocular disorders. In simulated cultures of proliferative intraocular disorders, cells decreased or retained their CK7, CK8, and CK18, retained Vim, and increased CK19 and GFAP, while their mesenchymal morphology became clearer over time.

CONCLUSIONS

Studies of intermediate filament proteins in vivo suggest that dedifferentiation occurs in RPE cells in ERM. Dedifferentiated RPE cells may be responsible for epithelioid-like and fibroblast/fusiform-like cells. Furthermore, changes in intermediate filament protein levels were observed in RPE cells in simulated cultures of proliferative intraocular disorders. These changes were linked to cells acquiring a mesenchymal migratory, phenotype. Results indicate that the dedifferentiation of RPE cells occurs both in vivo and in vitro and that it can be explained as an epithelial-mesenchymal transition.

Here we further characterize a number of properties inherent to the thermotolerant cell. In the preceding paper, we showed that the acquisition of the thermotolerant state (by a prior induction of the heat-shock proteins) renders cells translationally tolerant to a subsequent severe heat-shock treatment and thereby results in faster kinetics of both the synthesis and subsequent repression of the stress proteins. Because of the apparent integral role of the 70-kD stress proteins in the acquisition of tolerance, we compared the intracellular distribution of these proteins in both tolerant and nontolerant cells before and after a severe 45 degrees C/30-min shock. In both HeLa and rat embryo fibroblasts, the synthesis and migration of the major stress-induced 72-kD protein into the nucleolus and its subsequent exit was markedly faster in the tolerant cells as compared with the nontolerant cells. Migration of preexisting 72-kD into the nucleolus was shown to be dependent upon heat-shock treatment and independent of active heat-shock protein synthesis. Using both microinjection and immunological techniques, we observed that the constitutive and abundant 73-kD stress protein similarly showed a redistribution from the cytoplasm and nucleus into the nucleolus as a function of heat-shock treatment. We show also that other lesions that occur in cells after heat shock can be prevented or at least minimized if the cells are first made tolerant. Specifically, the heat-induced collapse of the intermediate filament cytoskeleton did not occur in cells rendered thermotolerant. Similarly, the disruption of intranuclear staining patterns of the small nuclear ribonucleoprotein complexes after heat-shock treatment was less apparent in tolerant cells exposed to a subsequent heat-shock treatment.

BHK-21 fibroblasts contain type III vimentin/desmin intermediate filament (IF) proteins that typically co-isolate and co-cycle in in vitro experiments with certain high molecular weight proteins. Here, we report purification of one of these and demonstrate that it is in fact the type VI IF protein nestin. Nestin is expressed in several fibroblastic but not epithelioid cell lines. We show that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein alpha-internexin to form heterodimer coiled-coil molecules. These molecules may co-assemble into 10 nm IF provided that the total amount of nestin does not exceed about 25%. However, nestin does not dimerize with types I/II keratin IF chains. The bulk of the nestin protein consists of a long carboxyl-terminal tail composed of various highly charged peptide repeats. By analogy with the larger neurofilament chains, we postulate that these sequences serve as cross-bridgers or spacers between IF and/or other cytoskeletal constituents. In this way, we propose that direct incorporation of modest amounts of nestin into the backbone of cytoplasmic types III and IV IFs affords a simple yet flexible method for the regulation of their dynamic supramolecular organization and function in cells.

Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated down-regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation.

The L55P transthyretin (TTR) familial amyloid polyneuropathy-associated variant is distinct from the other TTR variants studied to date and the wild-type protein in that the L55P tetramer can dissociate to the monomeric amyloidogenic intermediate and form fibril precursors under physiological conditions (pH 7.0, 37 degrees C). The activation barrier associated with L55P-TTR tetramer dissociation is lower than the barrier for wild-type transthyretin dissociation, which does not form fibrils under physiological conditions. The L55P-TTR tetramer is also very sensitive to acidic conditions, readily dissociating to form the monomeric amyloidogenic intermediate between pH 5.5-5.0 where the wild-type TTR adopts a nonamyloidogenic tetrameric structure. The formation of the L55P monomeric amyloidogenic intermediate involves subtle tertiary structural changes within the beta-sheet rich subunit as discerned from Trp fluorescence, circular dichroism analysis, and ANS binding studies. The assembly of the L55P-TTR amyloidogenic intermediate at physiological pH (pH 7.5) affords protofilaments that elongate with time. TEM studies suggest that the entropic barrier associated with filament assembly (amyloid fibril formation) is high in vitro, amyloid being defined by the laterally assembled four filament structure observed by Blake upon isolation of "fibrils" from the eye of a FAP patient. The L55P-TTR protofilaments formed in vitro bind Congo red and thioflavin T (albeit more weakly than the fibrils produced at acidic pH), suggesting that the structure observed probably represents an amyloid precursor. The structural continuum from misfolded monomer through protofilaments, filaments, and ultimately fibrils must be considered as a possible source of pathology associated with these diseases.

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament (IF) proteins that are expressed in glandular epithelia. Although the mechanism of keratin turnover is poorly understood, caspase-mediated degradation of type I keratins occurs during apoptosis and the proteasome pathway has been indirectly implicated in keratin turnover based on colocalization of keratin-ubiquitin antibody staining. Here we show that K8 and K18 are ubiquitinated based on cotransfection of His-tagged ubiquitin and human K8 and/or K18 cDNAs, followed by purification of ubiquitinated proteins and immunoblotting with keratin antibodies. Transfection of K8 or K18 alone yields higher levels of keratin ubiquitination as compared with cotransfection of K8/18, likely due to stabilization of the keratin heteropolymer. Most of the ubiquitinated species partition with the noncytosolic keratin fraction. Proteasome inhibition stabilizes K8 and K18 turnover, and is associated with accumulation of phosphorylated keratins, which indicates that although keratins are stable they still turnover. Analysis of K8 and K18 ubiquitination and degradation showed that K8 phosphorylation contributes to its stabilization. Our results provide direct evidence for K8 and K18 ubiquitination, in a phosphorylation modulated fashion, as a mechanism for regulating their turnover and suggest that other IF proteins could undergo similar regulation. These and other data offer a model that links keratin ubiquitination and hyperphosphorylation that, in turn, are associated with Mallory body deposits in a variety of liver diseases.

The presence of the intermediate filament protein nestin has been the predominant marker used to describe stem and progenitor cells in the mammalian CNS. In this study, a 998-bp fragment in the 3' region of the nestin mRNA was cloned from human fetal brain cells (HFBC). The nucleotide sequence of the cloned cDNA revealed 21 differences with the previously published human nestin sequence, resulting in 17 amino acid changes. A 150-amino-acid fragment derived from the cloned nestin cDNA was coupled to glutathione S-transferase and used as an immunogen to generate a rabbit polyclonal antiserum that selectively detects human nestin. HFBC that proliferated in response to basic fibroblast growth factor incorporated 5-bromo-2'-deoxyuridine into their nuclei and immunostained for nestin, indicating nestin expression in proliferating CNS progenitor cells. In all cell cultures, nestin costained with the neuroepithelial cell marker vimentin. A small subset of nestin-stained cells (1-2%) immunostained with neuronal marker MAP-2 during the first week and after 4 weeks in culture. However, during the first week in culture, approximately 10-30% of the total cell population of HFBC stained for the glial cell marker GFAP, and nearly all coimmunostained for nestin. After 4 weeks in culture, a subset of GFAP-positive cells emerged that no longer costained with nestin. These results describe nestin expression not only in CNS progenitor cells but also in the cells which were in transition from a progenitor stage to glial differentiation. Collectively, these data suggest a differential temporal regulation of nestin expression during glial and neuronal cell differentiation.

With retrovirus-mediated gene transfer, we used intact and deleted keratin proteins to investigate the molecular basis of intermediate filament function. Three levels of assembly show a different stringency for the involvement of individual keratin domains: protein accumulation requires the alpha helix domains; stable filament formation additionally requires both N- and C-terminal domains of either one of the two interacting keratins, suggesting that head to tail homotypic interaction is important for effective elongation; and higher order organization of the cytoplasmic network depends on correct type I-type II pairing of keratins. The presence of two distinct interaction sites along potentially different axes may explain the characteristic morphology of keratin intermediate filament networks.

Intermediate filaments (IFs) are abundant structures found in most eukaryotic cells, including those in the nervous system. In the CNS, the primary components of neuronal IFs are alpha-internexin and the neurofilament triplet proteins. In the peripheral nervous system, a fifth neuronal IF protein known as peripherin is also present. IFs in astrocytes are primarily composed of glial fibrillary acidic protein (GFAP), although vimentin is also expressed in immature astrocytes and some mature astrocytes. In this Review, we focus on the IFs of glial cells (primarily GFAP) and neurons as well as their relationship to different neurodegenerative diseases.

Multiple evidence suggests that fibroblast growth factors (FGFs), most prominently FGF-2, affect astroglial proliferation, maturation, and transition to a reactive phenotype in vitro, and after exogenous administration, in vivo. Whether this reflects a physiological role of endogenous FGF is unknown. Using FGF-2 and FGF-5 single- and double mutant mice we show now a region-specific reduction of glial fibrillary acidic protein (GFAP), but not of S100 in gray matter astrocytes. FGF-2 is apparently the major regulator of GFAP, because in mice deficient for FGF-2, GFAP is distinctly reduced in cortex and striatum, whereas in FGF-5-/- animals only a reduction in the midbrain tegmentum can be observed. In FGF-2-/-/FGF-5-/- double mutant animals, GFAP-immunoreactivity is reduced in all three brain regions. Cortical astrocytes cultured from FGF-2-/-/FGF-5-/- double mutant mice revealed reduced levels of GFAP, but not S100 as compared with wild-type littermates. This phenotype could be rescued by exogenous FGF-2 but not FGF-5 (10 ng/ml). Electron microscopy revealed reduced levels of intermediate filaments in perivascular astroglial endfeet. This defect was accompanied by enhanced permeability of the blood-brain barrier (BBB), as detected by albumin extravasation. Levels of the tight junction proteins Occludin and ZO-1 were reduced in blood vessels of FGF-2-/-/FGF-5-/- double mutant mice as compared with wild-type littermates. Our data support the notion that endogenous FGF-2 and FGF-5 regulate GFAP expression in a region-specific manner. The observed defect in astroglial differentiation is accompanied by a defect in BBB function arguing for an indirect or direct role of FGFs in the regulation of BBB permeability in vivo.

Transgenic approaches have recently been used to investigate the functions of neuronal intermediate filaments. Gene knockout studies have demonstrated that neurofilaments are not required for axogenesis and that individual neurofilament proteins play distinct roles in filament assembly and in the radial growth of axons. The involvement of neurofilaments in disease is supported by the discovery of novel mutations in the neurofilament heavy gene from cases of amyotrophic lateral sclerosis and by reports of neuronal death in mouse models expressing neurofilament and alpha-internexin transgenes. However, mouse studies have shown that axonal neurofilaments are not required for pathogenesis caused by mutations in superoxide dismutase and that increasing perikaryal levels of neurofilament proteins may even confer protection in this disease.

Intermediate filaments (IFs) are assembled from a diverse group of evolutionarily conserved proteins and are specified in a tissue-dependent, cell type-dependent, and context-dependent fashion in the body. IFs are involved in multiple cellular processes that are crucial for the maintenance of cell and tissue integrity and the response and adaptation to various stresses, as conveyed by the broad array of crippling clinical disorders caused by inherited mutations in IF coding sequences. Accordingly, the expression, assembly, and organization of IFs are tightly regulated. Migration is a fitting example of a cell-based phenomenon in which IFs participate as both effectors and regulators. With a particular focus on vimentin and keratin, we here review how the contributions of IFs to the cell's mechanical properties, to cytoarchitecture and adhesion, and to regulatory pathways collectively exert a significant impact on cell migration.

Lamins are karyoskeletal proteins associated with the nuclear envelope which can be divided into two groups, i.e. the type A lamins of near neutral pI and the more acidic lamins, including mammalian lamin B. We have isolated cDNA clones encoding a representative of the type B subfamily from Xenopus laevis, and have deduced its amino acid sequence from the coding portion of the approximately 2.9 kb mRNA. The polypeptide (mol. wt 66,433) is identified as a typical lamin by its homology to Xenopus human type A lamins, but detailed sequence comparison shows that LI is less related to Xenopus lamin A than the latter is to human lamin A. The conformation predicted for LI conforms to the general model of lamins and intermediate filament proteins and is characterized by an extended central alpha-helical coiled coil domain, flanked by non-alpha-helical domains, i.e. a relatively short N-terminal head and a long C-terminal tail. As in lamins A and C, the head of lamin LI is positively charged and the tail presents a similar C-terminal pentapeptide, a putative nuclear accumulation signal, a very negatively charged region and a number of short regions that are highly homologous in all lamins. However, LI differs from the type A lamins by the absence of the oligo-histidine stretch and a di-proline motif in the tail region and by a significantly lower number of identical amino acid positions.(ABSTRACT TRUNCATED AT 250 WORDS)

Certain smooth muscle cells of blood vessel walls as well as cultured baby hamster kidney cells contain simultaneously two different intermediate-sized filament (IF) proteins, desmin and vimentin. We have examined the question of the occurrence of both proteins in the same IF by chemically crosslinking the single cysteine group present in each of them. Oxidative crosslinking of filaments present in cytoskeletal preparations with cupric ion complexes of 1,10-phenanthroline resulted in formation of three types of dimers: vimentin-vimentin, desmin-desmin, and vimentin-desmin. These dimers were separated by NaDodSO4/polyacrylamide gel electrophoresis and characterized by binding of specific antibodies, by one- and two-dimensional gel electrophoresis of monomers obtained after cleavage of the disulfide bond by thiol agents, and by mapping of radioiodinated tryptic peptides. The demonstration of heterodimers of vimentin and desmin in vascular smooth muscle tissue of cow and chicken and in baby hamster kidney cells shows that the two proteins can be integrated in the same IF and can be nearest neighbors, oriented with their cysteine residues in a mirror-image symmetry. The possible existence of heteropolymer IF in other cell types is discussed.

Abundant abnormal aggregates of cytoskeletal proteins are neuropathological signatures of many neurodegenerative diseases that are broadly classified by filamentous aggregates of neuronal intermediate filament (IF) proteins, or by inclusions containing the microtubule-associated protein (MAP) tau. The discovery of mutations in neuronal IF and tau genes firmly establishes the importance of neuronal IF proteins and tau in the pathogenesis of neurodegenerative diseases. Multiple IF gene mutations are pathogenic for Charcot-Marie-Tooth (CMT) disease and amyotrophic lateral sclerosis (ALS)--in addition to those in the copper/zinc superoxide dismutase-1 (SOD1) gene. Tau gene mutations are pathogenic for frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), and tau polymorphisms are genetic risk factors for sporadic progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Thus, IF and tau abnormalities are linked directly to the aetiology and pathogenesis of neurodegenerative diseases. In vitro and transgenic animal models are being used to demonstrate that different mutations impair protein function, promote tau fibrilization, or perturb tau gene splicing, leading to aberrant and distinct tau aggregates. For recognition of these disorders at neuropathological examination, immunohistochemistry is needed, and this may be combined with biochemistry and molecular genetics to properly determine the nosology of a particular case. As reviewed here, the identification of molecular genetic defects and biochemical alterations in cytoskeletal proteins of human neurodegenerative diseases has facilitated experimental studies and will promote the development of assays of molecules which inhibit abnormal neuronal IF and tau protein inclusions.

Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 "anti-recombinase" restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we cytologically characterize Srs2 function in vivo and describe a novel mechanism for regulating the initiation of HR. We find that Srs2 is recruited separately to replication and repair centers and identify the genetic requirements for recruitment. In the absence of Srs2 activity, Rad51 foci accumulate, and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2 removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR.

Keratin intermediate filament proteins form cytoskeletal scaffolds in epithelia, the disruption of which affects cytoarchitecture, cell growth, survival, and organelle transport. However, owing to redundancy, the global function of keratins has not been defined in full. Using a targeted gene deletion strategy, we generated transgenic mice lacking the entire keratin multiprotein family. In this study, we report that without keratins, embryonic epithelia suffer no cytolysis and maintain apical polarity but display mislocalized desmosomes. All keratin-null embryos die from severe growth retardation at embryonic day 9.5. We find that GLUT1 and -3 are mislocalized from the apical plasma membrane in embryonic epithelia, which subsequently activates the energy sensor adenosine monophosphate kinase (AMPK). Analysis of the mammalian target of rapamycin (mTOR) pathway reveals that AMPK induction activates Raptor, repressing protein biosynthesis through mTORC1's downstream targets S6 kinase and 4E-binding protein 1. Our findings demonstrate a novel keratin function upstream of mTOR signaling via GLUT localization and have implications for pathomechanisms and therapy approaches for keratin disorders and the analysis of other gene families.

Investigations into the biological actions of nerve growth factor (NGF) have shown that dorsal root ganglion (DRG) neurons subserving nociception require NGF for survival and maintenance of phenotype. This discovery suggests that the signaling NGF receptor, TrkA, can be used as a marker for nociceptive neurons. In this study, we have used antibodies to TrkA, in conjunction with cell biological markers that show a restricted distribution in the DRG, to further characterize subsets of DRG neurons that are dependent upon NGF. Staining for TrkA labeled small and medium-sized neurons that composed 47% of all neurons in thoracic ganglia. Double-labeling with antibodies to the high molecular weight neurofilament protein (NFH), a marker for neurons with myelinated axons, demonstrated that TrkA staining is found in only a small subset of myelinated neurons. Surprisingly, many DRG neurons were not labeled by either TrkA or NFH. These neurons had small soma areas, contained the intermediate filament protein peripherin, and were labeled by the lectin BSI, identifying them as neurons likely to have unmyelinated axons. In addition, small TrkA-negative neurons were extensively labeled by antibodies to the intermediate filament protein alpha-internexin, the delta isoform of protein kinase C, and by the BSI isolectin BSI-B4. In order to assess the potential functions of TrkA-negative small neurons, we examined their projections to the dorsal horn of the spinal cord. TrkA-immunoreactivity in the spinal cord was restricted to lamina I and the outer region of lamina II (IIo), similar to staining for calcitonin gene-related peptide. In contrast, the central projections of TrkA-negative neurons, as visualized by BSI-B4 staining, were particularly dense in lamina IIi. Our results suggest that TrkA-expressing and non-TrkA-expressing small neurons compose functionally distinct populations of DRG neurons.

Peripherin, a type III intermediate filament (IF) protein, upregulated by injury and inflammatory cytokines, is a component of IF inclusion bodies associated with degenerating motor neurons in sporadic amyotrophic lateral sclerosis (ALS). We report here that sustained overexpression of wild-type peripherin in mice provokes massive and selective degeneration of motor axons during aging. Remarkably, the onset of peripherin-mediated disease was precipitated by a deficiency of neurofilament light (NF-L) protein, a phenomenon associated with sporadic ALS. In NF-L null mice, the overexpression of peripherin led to early- onset formation of IF inclusions and to the selective death of spinal motor neurons at 6 mo of age. We also report the formation of similar peripherin inclusions in presymptomatic transgenic mice expressing a mutant form of superoxide dismutase linked to ALS. Taken together, these results suggest that IF inclusions containing peripherin may play a contributory role in motor neuron disease.

Epithelial cells contain a class of intermediate-sized filaments formed by proteins related to epidermal alpha-keratins ('cytokeratins'). Different epithelia can express different combinations of cytokeratin polypeptides widely varying in apparent mol. wt. (40 000-68 000) and isoelectric pH (5.0-8.5). We have separated, by two-dimensional gel electrophoresis, cytokeratin polypeptides from various tissues and cultured cells of man, cow, and rodents and examined their relatedness by tryptic peptide mapping. By this method, a subfamily of closely related cytokeratin polypeptides has been identified which comprises the relatively large (greater than or equal to mol. wt. 52 500 in human cells) and basic (pH greater than or equal to 6.0) polypeptides but not the smaller and acidic cytokeratins. In all species examined, the smallest polypeptide of this subfamily is cytokeratin A, which is widespread in many simple epithelia and is the first cytokeratin expressed during embryogenesis. This cytokeratin polypeptide subfamily is represented by at least one member in all epithelial and carcinoma cells examined, indicating that polypeptides of this subfamily serve an important role as tonofilament constitutents . Diverse stratified epithelia and tumours derived therefrom contain two or more polypeptides of this subfamily, and the patterns of expression in different cell types suggest that some polypeptides of this subfamily are specific for certain routes of epithelial differentiation.

Keratins K5 and K14 form the extensive intermediate filament network of mitotically active basal cells in all stratified epithelia. We have explored the regulatory mechanisms governing cell-type-specific and differentiation stage-specific expression of the human K5 gene in transiently transfected keratinocytes in vitro and in transgenic mice in vivo. Six thousand base pairs of 5' upstream K5 sequence directed proper basal cell-specific expression in all stratified epithelia. Surprisingly, as few as 90 bp of the K5 promoter still directed expression to stratified epithelia, with expression predominantly in epidermis, hair follicles, and tongue. Despite keratinocyte-preferred expression, the truncated K5 promoter displayed departures from basal to suprabasal expression in epidermis and from outer root sheath to inner root sheath expression in the follicle, with some regional variations in expression as well. To begin to elucidate the molecular controls underlying the keratinocyte specificity of the truncated promoter, we examined protein-DNA interactions within this region. A number of keratinocyte nuclear proteins bind to a K5 gene segment extending from -90 to +32 bp and are functionally involved in transcriptional regulation in vitro. Interestingly, several of these factors are common to both the K5 and K14 promoters, although they appear to be distinct from those previously implicated in keratinocyte specificity. Mutagenesis studies indicate that factors binding in the vicinity of the TATA box and transcription initiation are responsible for the cell type specificity of the truncated K5 promoter.

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.