Cytokeratin domains involved in heterotypic complex formation determined by in-vitro binding assays

Cytokeratins are constituent proteins of intermediate filaments (IFs) that form heterotypic tetrameric IF subunits containing two polypeptide chains of each of the two cytokeratin subfamilies, i.e. the acidic (type I) and the basic (type II). To locate the molecular domains involved in the formation of these heterotypic complexes, we have developed a binding assay in which total cellular or cytoskeletal polypeptides, or proteolytically prepared cytokeratin fragments, are separated by one-, or two-dimensional gel electrophoresis, blot-transferred on to nitrocellulose paper and probed with radio-iodinated purified cytokeratin polypeptides or fragments thereof, using buffers of various ionic strengths with or without 4 M-urea. Using these polypeptides in the binding assay, specific heterotypic binding was observed between complementary cytokeratin polypeptides of the two subfamilies (but not with other IF proteins) and between the corresponding alpha-helical rod domain fragments. Both rod coils 1 and 2 of the type II cytokeratin 8 bound to the rod (coils 1 and 2) fragment of type I cytokeratins, and this binding occurred at both low and high ionic strengths. The results obtained indicate that: (1) the binding between cytokeratin polypeptides of the complementary type is stronger and more selective than interactions of cytokeratins with other IF and non-IF proteins; (2) both the head and the tail portions of the proteins are not required for heterotypic complex formation; (3) the complementarity information located in the alpha-helical portions of the rod domain, and in short sequences immediately flanking them, is sufficient to discriminate between the two types of cytokeratins and to secure the formation of heterotypic cytokeratin complexes; (4) both coils 1 and 2 of the rod can contribute to this association; and (5) the formation of the heterotypic cytokeratin complex is not critically dependent upon ionic interactions. Our results are further compatible with the concept that the heterotypic binding takes place between cytokeratin homodimer coiled-coils.     

The complement of native α-keratin polypeptides of hair-forming cells: A subset of eight polypeptides that differ from epithelial cytokeratins

Living hair-forming cells (trichocytes) were obtained from basal portions of human, bovine and ovine hair-follicles, free from contaminations of root-sheath epithelia. Their intermediate filament (IF) cytoskeleton was studied by gel electrophoresis of the native, i.e. non-S-carboxymethylated polypeptides, by peptide-map analysis of the individual components, by reconstitution experiments and by immunological methods. The IF protein complement of trichocytes from all three species is characterized by a very similar set of eight highly conserved alpha-keratin polypeptides, comprising four members of the basic (type II; Mr 56,500-60,000) and four members of the acidic (type I; Mr 41,000-44,000) cytokeratin subfamily. None of these eight trichocyte alpha-keratin polypeptides, which form heterotypic complexes and IF in vivo and in vitro, is identical to any of the epithelial cytokeratins of the same species. All the trichocyte-specific cytokeratins are native polypeptides encoded by different mRNAs, as demonstrated by in vitro translation of hair follicle mRNA. The same polypeptides are also found in mature hairs, although with different patterns of modification. Our study provides the first analysis of the native unmodified alpha-keratin polypeptides of trichocytes and hairs and therefore allows a direct comparison of these with the epithelial cytokeratins and other IF proteins from the same species. These findings indicate that, during fetal hair-follicle formation, the differentiation of trichocytes from epithelial cells involves a complete cessation of the synthesis of epithelial cytokeratins and a marked induction of the synthesis of a complex set of trichocyte-specific cytokeratins.     

De novo formation of cytokeratin filaments in calf lens cells and cytoplasts after transfection with cDNAs or microinjection with mRNAs encoding human cytokeratins.

We have studied the formation of new intermediate-sized filaments (IFs) by human cytokeratins (CKs) 8 and/or 19 in cultured bovine lens cells stably transfected with the corresponding cDNAs under SV40 promoter control. In the transfected cells, polypeptides of both type I and type II CKs were synthesized to near-equimolar amounts, formed heterotypic complexes and assembled into IFs with a peculiar tendency to accumulate into variously sized, often roundish aggregates in the juxtanuclear region, usually one per cell. Electron microscopy of these large CK IF aggregates revealed typical 7 to 12-nm IFs, tightly packed together in an apparently haphazard mode. By immunoelectron microscopy, the CK IFs could be readily distinguished from the vimentin IFs which were abundant in these cells. Electron microscopy also showed that many of the CK IF aggregates were located in the vicinity of the nucleus but did not have direct contact with the nuclear envelope; moreover, their location did not regularly correspond to those of the centrosomes and the Golgi apparatus. During enucleation of transfected cells in the presence of cytochalasin B, the CK aggregates were often retained in the cytoplast. After microinjection of CK 8 and 19 mRNAs, synthesized in vitro from cDNA molecules, into enucleated cytoplasts prepared from untransfected cells, CK IFs similar to those observed in microinjected whole cells were formed but often showed a wider cytoplasmic distribution. Our observations indicate that typical CK IFs can form, in vivo, in the absence of any nuclear structures. We discuss possible reasons for the tendency of the CK IFs to accumulate, in this cell line, into a juxtanuclear aggregate, in relation to similar CK-IF aggregates formed in certain normal cell types and upon toxic damages.     

Monoclonal cytokeratin antibody recognizing a heterotypic complex: immunological probing of conformational states of cytoskeletal proteins in filaments and in solution

A novel type of monoclonal murine antibody (Ks18.18) directed against an epitope depending on human cytokeratin (CK) 18, a member of the acidic (type I) CK subfamily, is described. We show by SDS-PAGE immunoblots and dot-blot assays that this antibody is unreactive with both the denatured and the renatured individual polypeptides but binds strongly to heterotypic coiled-coil complexes of CK 18 with several members of the complementary basic (type II) CK subfamily, notably with CK 8; i.e., its most frequent natural partner. We also show that specific interactions between complementary CK polypeptides take place during the incubation steps of immunoblotting procedures as polypeptides, or fragments thereof, that detach from the substrate can bind to complementary polypeptides attached to the substratum, which may result in false assignments of antibody reactivities. The conformation-specific, CK 18-dependent epitope of Ks18.18 was detected in intermediate filaments (IFs) of cultured cells, simple epithelia, and many carcinomas and, surprisingly, also in the basal cells of some stratified epithelia. Ks18.18 also reacts with altered CK configurations as present in the spheroidal bodies of mitotic cells and in the Mallory bodies of hepatocytes intoxicated with certain drugs, thus indicating that the heterotypic CK complexes are maintained in these structures. We have also used antibody Ks18.18 to demonstrate the existence of heterotypic CK 8 and 18 complexes in a distinct soluble form among supernatant proteins from cell homogenates which is indistinguishable from the heterotypic tetramer obtained after experimental disintegration of IFs. The potential value of such IF conformation-specific antibodies in cell biological research and pathology is discussed.     

Analysis of cytokeratin domains by cloning and expression of intact and deleted polypeptides in Escherichia coli.

Using recombination of an appropriate expression vector system (pINDU) with a complete cDNA encoding a basic (type II) cytokeratin, i.e. cytokeratin 8 (1) of Xenopus laevis, we transformed Escherichia coli cells to synthesize considerable amounts of an insoluble eukaryotic cytoskeletal protein. The cytokeratin was deposited in large 'inclusion bodies' in the bacterial cytoplasm but did not form detectable filamentous structures. However, when the E. coli-expressed cytokeratin was purified and combined in vitro with an authentic cytokeratin of the complementary, i.e. acidic (type I) subfamily, it formed typical intermediate-sized filaments (IFs). Using Bal31 deletion from either the 5' or the 3' end of the cDNA, series of polypeptides progressively deleted from the amino or the carboxy terminus were produced in E. coli and identified by monoclonal antibodies. These assays allowed the mapping of epitopes. The deletion polypeptides of cytokeratin 8 were further examined to localize the region(s) involved in the heterotypic binding of alpha-helices of type I cytokeratins, using an in vitro nitrocellulose blot binding assay. We show that a region of 37 amino acids located in the central portion of coil 2 of the alpha-helical rod domain is sufficient for the specific recognition of a radiolabelled type I cytokeratin, i.e. cytokeratin 18 (D) from rat liver. In addition, deletion polypeptides containing only coil 1 of the alpha-helical rod also bind strongly the complementary cytokeratin. This indicates that the capability of heterotypic recognition and complex formation is not restricted to a single signal sequence but is located in distant and independent alpha-helical domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentially expressed bovine cytokeratin genes. Analysis of gene linkage and evolutionary conservation of 5''-upstream sequences.

Cytokeratins are a family of approximately 20 polypeptides which form the intermediate-sized filaments (IFs) characteristic of epithelial cells. They are synthesized co-ordinately as 'pairs' consisting of one representative from each of the two cytokeratin subfamilies, i.e. the acidic (type I) and the more basic (type II) polypeptides, in cell type-specific combinations. We have isolated and characterized the genes coding for four bovine cytokeratins of the basic (type II) subfamily, i.e. cytokeratins Ib, III, IV and 6*, by Southern blot hybridization, hybridization-selection-translation experiments, hetero-duplex mapping, and partial sequencing of the exons coding for the hypervariable carboxy-terminal 'tail' regions of the proteins and the 3'-non-translated ends of the mRNAs which are distinct for the individual cytokeratin polypeptides. Limited 'chromosomal walk' experiments demonstrated that the genes are organized into two tandems, i.e. 6*----Ib and III----IV, in which they are separated by approximately 11 kb. RNA analysis by Northern and dot blots show that both genes of the III----IV tandem are co-expressed in some bovine tissues (muzzle epidermis, hoof pad and tongue mucosa) and cultured cells (BMGE + H) but that in other tissues, cornea for example, only the gene encoding III is expressed. Unexpectedly, the genes linked in the tandem 6*----Ib are not co-expressed in any of the tissues examined. mRNA from gene 6* has been found in tongue mucosa but in none of the other cell lines and tissues examined, whereas mRNA for cytokeratin Ib is expressed in cornea and muzzle epidermis but not in, for example, tongue mucosa and in the epidermis of the heel pad.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-expression of cytokeratins and neurofilament proteins in a permanent cell line: cultured rat PC12 cells combine neuronal and epithelial features

The cytoskeleton of the rat cultured cell line PC12, which is widely used in cell biology as a model system for neuron-like differentiation, displays an unusual combination of intermediate-sized filaments (IFs). As determined by electron microscopy, immunolocalization, and biochemical analyses, these cells contain, in addition to neurofilaments, an extended meshwork of bundles of cytokeratin IFs comprising cytokeratins A and D, equivalent to human cytokeratin polypeptides Nos. 8 and 18, irrespective of whether they are grown in the presence or absence of nerve growth factor. The two IF systems differ in their fibrillar arrays, the neurofilaments being concentrated in perinuclear aggregates similar to those found in certain neuroendocrine tumors of epithelial origin. We conclude that PC12 cells permanently co-express IFs of both the epithelial and the neuronal type and thus present an IF combination different from those of adrenal medulla cells and pheochromocytomas, i.e., the putative cells of origin of the line PC12. The IF cytoskeleton of PC12 cells resembles that of various neuroendocrine tumors derived from epithelial cells. The results show that the development of a number of typical neuronal differentiation features is compatible with the existence of an epithelial type IF cytoskeleton, i.e., cytokeratins. The implications of these findings concerning the validity of the PC12 cell line as a model for neuronal differentiation and possible explanations of the origin of cells with this type of IF co-expression are discussed.     

Low level expression of cytokeratins 8, 18 and 19 in vascular smooth muscle cells of human umbilical cord and in cultured cells derived therefrom, with an analysis of the chromosomal locus containing the cytokeratin 19 gene

Of the various intermediate filament (IF) proteins certain cytokeratins, usually a hallmark of epithelial differentiation, can also be detected in some non-epithelial cells in low amounts. We have studied a representative case of this atypical expression, the smooth muscle cells of the blood vessel walls of the human umbilical cord, at the protein and nucleic acid level, by light and electron microscopic immunolocalization, gel electrophoresis and immunoblotting of cytoskeletal proteins, and mRNA identification by Northern blotting. For the latter we have used sensitive probes for various cytokeratins, including new probes for cytokeratin 19. We also describe the chromosome 17 locus comprising the genes for cytokeratins 15 and 19, and we emphasize the occurrence of several unusual and evolutionarily stable sequence elements in the introns of the cytokeratin 19 gene. Most, perhaps all smooth muscle cells of these blood vessels, positively identified by the presence of desmin and smooth muscle type alpha-actin, are immunostained by antibodies specific for cytokeratins 8 and 18, and a subpopulation also contains cytokeratin 19. Immunoelectron microscopy indicates that these cytokeratins are arranged in IFs that are distributed differently from the majority of the IFs formed by desmin and vimentin. Gel electrophoresis of cytoskeletal proteins from microdissected vascular wall tissue shows that the amounts of cytokeratins 8 and 18 present in these tissues are very low, representing less than 1% of the total IF protein, and that cytokeratin 19 is present only in trace amounts. Correspondingly, the contents of mRNAs for cytokeratins 8, 18 and 19 in these tissues are much lower than those present in epithelial cells examined in parallel. We have also established cell cultures derived from umbilical cord vascular smooth muscles that have maintained the expression of cytokeratins 8, 18 and 19, together with vimentin and the smooth muscle type alpha-actin, but do not synthesize desmin. In these cell cultures the cytokeratins are present in much higher amounts than in the original tissue and form IFs that, surprisingly, show a similar distribution as the vimentin IFs and, upon treatment of the cells with colcemid, collapse into juxtanuclear aggregates, often even more effectively than the vimentin IFs do. We conclude that in a certain subtype of smooth muscle cells, the genes encoding cytokeratins of the "simple epithelial type", i.e., cytokeratins 8, 18 and 19, are expressed and that the low level expression of these genes is compatible with myogenic differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

An epithelium-type cytoskeleton in a glial cell: astrocytes of amphibian optic nerves contain cytokeratin filaments and are connected by desmosomes

In higher vertebrates the cytoskeleton of glial cells, notably astrocytes, is characterized (a) by masses of intermediate filaments (IFs) that contain the hallmark protein of glial differentiation, the glial filament protein (GFP); and (b) by the absence of cytokeratin IFs and IF-anchoring membrane domains of the desmosome type. Here we report that in certain amphibian species (Xenopus laevis, Rana ridibunda, and Pleurodeles waltlii) the astrocytes of the optic nerve contain a completely different type of cytoskeleton. In immunofluorescence microscopy using antibodies specific for different IF and desmosomal proteins, the astrocytes of this nerve are positive for cytokeratins and desmoplakins; by electron microscopy these reactions could be correlated to IF bundles and desmosomes. By gel electrophoresis of cytoskeletal proteins, combined with immunoblotting, we demonstrate the cytokeratinous nature of the major IF proteins of these astroglial cells, comprising at least three major cytokeratins. In this tissue we have not detected a major IF protein that could correspond to GFP. In contrast, cytokeratin IFs and desmosomes have not been detected in the glial cells of brain and spinal cord or in certain peripheral nerves, such as the sciatic nerve. These results provide an example of the formation of a cytokeratin cytoskeleton in the context of a nonepithelial differentiation program. They further show that glial differentiation and functions, commonly correlated with the formation of GFP filaments, are not necessarily dependent on GFP but can also be achieved with structures typical of epithelial differentiation; i.e., cytokeratin IFs and desmosomes. We discuss the cytoskeletal differences of glial cells in different kinds of nerves in the same animal, with special emphasis on the optic nerve of lower vertebrates as a widely studied model system of glial development and nerve regeneration.     

Spontaneous losses of control of cytokeratin gene expression in transformed, non-epithelial human cells occurring at different levels of regulation

Intermediate filaments (IFs) of the cytokeratin (CK) type are cytoskeletal elements typical for epithelial differentiation. However, in diverse transformed culture lines of non-epithelial origin, rare cells emerge spontaneously, which synthesize, in addition to their vimentin IFs, CKs 8 and 18. We enriched such cells by cloning and studied the level(s) of regulation at which these changes occur. We found that in SV40-transformed fibroblasts the CK 18 gene is constitutively transcribed into translatable mRNA but that the protein is rapidly degraded in the absence of its complex partner, CK 8. In contrast, cells immunocytochemically positive for CK IFs contained both CKs 8 and 18, which apparently stabilized in heterotypic complexes. These findings and related observations of active genes for CKs 8 and/or 18 in several other transformed non-epithelial cell lines indicate that the genes for CKs 18 and, less frequently, 8 can be active in diverse different non-epithelial cell lines; synthesis of type I and type II CK pair partners can be uncoupled; control of CK IF formation can take place at different levels. We suggest that the intrinsic instability of the inactive state of these genes is responsible for the occurrence of CKs 8 and 18 in certain non-epithelial tissues and tumors, a caveat in tumor diagnosis.     

Complete sequence of a bovine type I cytokeratin gene: conserved and variable intron positions in genes of polypeptides of the same cytokeratin subfamily

The complete sequence of a bovine gene encoding an epidermal cytokeratin of mol. wt. 54 500 (No VIb) of the acidic (type I) subfamily is presented, including an extended 5' upstream region. The gene (4377 bp, seven introns) which codes for a representative of the glycine-rich subtype of cytokeratins of this subfamily, is compared with genes coding for: another subtype of type I cytokeratin; a basic (type II) cytokeratin gene; and vimentin, a representative of another intermediate filament (IF) protein class. The positions of the five introns located within the highly homologous alpha-helix-rich rod domain are identical or equivalent, i.e., within the same triplet, in the two cytokeratin I genes. Four of these intron positions are also identical with intron sites in the vimentin gene, and three of these intron positions are identical or similar in the type I and type II cytokeratin subfamilies. On the other hand, the gene organization of both type I cytokeratins differs from that of the type II cytokeratin in the rod region in five intron positions and in the introns located in the carboxy-terminal tail region, with the exception of one position at the rod-tail junction. Remarkably, the two type I cytokeratins also differ from each other in the positions of two introns located at and in the region coding for the hypervariable, carboxy-terminal portion. The introns and the 5' upstream regions of the cytokeratin VIb gene do not display notable sequence homologies with the other IF protein genes, but sequences identical with--or very similar to--certain viral and immunoglobulin enhancers have been identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues. I. Human and bovine hair follicles

The cytokeratin family of intermediate filament (IF) proteins can be grouped into the epithelial polypeptides ("soft alpha-keratins"), of which at least 19 exist in the various human epithelia, and the hair-type cytokeratins ("hard alpha-keratins"), which are typical of trichocytes, i.e., the living hair-forming cells. We have recently shown [34] that the hair follicles from diverse mammalian species contain a set of eight major cytokeratin polypeptides, four each of the acidic (type I) and the basic (type II) subfamily, which are different from all known epithelial cytokeratins. In addition, we have identified two new minor trichocytic cytokeratin polypeptides, designated Hax (type I) and Hbx (type II). Antibodies against trichocytic cytokeratins that do not crossreact with any of the epithelial cytokeratins have enabled us to study the expression of both kinds of cytokeratin in the various cell types of human and bovine hair follicles. Using immunofluorescence microscopy, we have observed intense reactions of trichocytic cytokeratins only in cells contributing to the forming hairs, i.e., hair shaft, medulla and cuticle, whereas immunostaining of the peribulbar matrix cells was weaker, if at all detectable. In contrast, epithelial cytokeratins were localized in both the inner and outer root sheath epithelia but, surprisingly, also in certain portions of the trichocyte column, notably cells of the cuticle, certain medullary cells, and trichocytes of the basalmost peripapillary cell layers. Cells coexpressing trichocytic and epithelial cytokeratins have been identified by double-label immunofluorescence microscopy. Epithelial cytokeratins of the inner and outer root sheath epithelia include, most remarkably, "simple-epithelium-type" cytokeratins 8, 18, and 19; these occur in certain peribulbar regions, in distinct patterns, but with variable frequencies. The occurrence of simple epithelial cytokeratins in hair follicles has also been confirmed by high-sensitivity immunoblotting of follicular polypeptides separated by gel electrophoresis. Vimentin-positive cells were abundantly interspersed (in some follicles, but not in all) between the trichocytes of the peripapillary cone, most of them probably being melanocytes. The cell-type complexity of the hair follicle and the different patterns of cytoskeletal protein expression in the various hair follicle cells are discussed in relation to the development and growth of this organ.     

Chromosomal assignments of human type I and type II cytokeratin genes to different chromosomes

The chromosomal location of representative members of the type I and type II subfamilies of the cytokeratin multigene family was determined using specific cDNA probes in Southern blot hybridization with DNA from somatic cell hybrids. Our results show that the gene encoding human type II cytokeratin 4 resides on chromosome 12 and that encoding type I cytokeratin 15 is located on chromosome 17. The results indicate that cytokeratins are not concentrated in only one cluster. The possibility of the existence of separate type I and type II cytokeratin gene clusters is discussed.     

Molecular characterization and expression of the stratification-related cytokeratins 4 and 15

A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oligonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratification-related cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed.     

The 47-kD lens-specific protein phakinin is a tailless intermediate filament protein and an assembly partner of filensin

In previous studies we have characterized a lens-specific intermediate filament (IF) protein, termed filensin. Filensin does not self-assemble into regular IFs but is known to associate with another 47-kD lens- specific protein which has been suggested to represent its assembly partner. To address this possibility, we cloned and sequenced the cDNA coding for the bovine 47-kD protein which we have termed phakinin (from the greek phi alpha kappa omicron sigma = phakos = lens). The predicted sequence comprises 406 amino acids and shows significant similarity (31.3% identity over 358 residues) to type I cytokeratins. Phakinin possesses a 95-residue, non-helical domain (head) and a 311 amino acid long alpha-helical domain punctuated with heptad repeats (rod). Similar to cytokeratin 19, phakinin lacks a COOH-terminal tail domain and it therefore represents the second known example of a naturally tailless IF protein. Confocal microscopy on frozen lens sections reveals that phakinin colocalizes with filensin and is distributed along the periphery of the lens fiber cells. Quantitative immunoblotting with whole lens fiber cell preparations and fractions of washed lens membranes suggest that the natural stoichiometry of phakinin to filensin is approximately 3:1. Under in vitro conditions, phakinin self- assembles into metastable filamentous structures which tend to aggregate into thick bundles. However, mixing of phakinin and filensin at an optimal ratio of 3:1 yields stable 10-nm filaments which have a smooth surface and are ultrastructurally indistinguishable from "mainstream" IFs. Immunolabeling with specific antibodies shows that these filaments represent phakinin/filensin heteropolymers. Despite its homology to the cytokeratins, phakinin does not coassemble with acidic (type I), or basic (type II) cytokeratins. From these data we conclude that filensin and phakinin are obligate heteropolymers which constitute a new membrane-associated, lens-specific filament system related to, but distinct from the known classes of IFs.     

Amino acid sequence and gene organization of cytokeratin no. 19, an exceptional tail-less intermediate filament protein.

We have isolated a cDNA clone from a bovine bladder urothelium library which encodes the smallest intermediate filament (IF) protein known, i.e. the simple epithelial cytokeratin (equivalent to human cytokeratin 19) previously thought to have mol. wt 40,000. This clone was then used to isolate the corresponding gene from which we have determined the complete nucleotide sequence and deduced the amino acid sequence of the encoded protein. This cytokeratin of 399 amino acids (mol. wt 43,893) is identified as a typical acidic (type I) cytokeratin but differs from all other IF proteins in that it does not show the carboxyterminal, non-alpha-helical tail domain. Instead it contains a 13 amino acids extension of the alpha-helical rod. The gene encoding cytokeratin 19 is also exceptional. It contains only five introns which occur in positions corresponding to intron positions in other IF protein genes. However, an intron which in all other IF proteins demarcates the region corresponding to the transition from the alpha-helical rod into the non-alpha-helical tail is missing in the cytokeratin 19 gene. Using in vitro reconstitution of purified cytokeratin 19 we show that it reacts like other type I cytokeratins in that it does not form, in the absence of a type II cytokeratin partner, typical IF. Instead it forms 40-90 nm rods of 10-11 nm diameter which appear to represent lateral associations of a number of cytokeratin molecules. Our results demonstrate that the non-alpha-helical tail domain is not an indispensable feature of IF proteins. The gene structure of this protein provides a remarkable case of a correlation of a change in protein conformation with an exon boundary.     

Mutational analysis of human papillomavirus E4 proteins: identification of structural features important in the formation of cytoplasmic E4/cytokeratin networks in epithelial cells.

We have previously demonstrated that human papillomavirus type 1 (HPV 1) and 16 (HPV 16) E4 proteins form cytoplasmic filamentous networks which specifically colocalize with cytokeratin intermediate-filament (IF) networks when expressed in simian virus 40-transformed keratinocytes. The HPV 16 (but not the HPV 1) E4 protein induced the collapse of the cytokeratin networks. (S. Roberts, I. Ashmole, G. D. Johnson, J. W. Kreider, and P. H. Gallimore, Virology 197:176-187, 1993). The mode of interaction of E4 with the cytokeratin IFs is unknown. To identify E4 sequences important in mediating this interaction, we have constructed a large panel of mutant HPV (primarily HPV 1) E4 proteins and expressed them by using the same simian virus 40-epithelial expression system. Mutation of HPV 1 E4 residues 10 to 14 (LLGLL) abrogated the formation of cytoplasmic filamentous networks. This sequence corresponds to a conserved motif, LLXLL, found at the N terminus of other E4 proteins, and similar results were obtained on deletion of the HPV 16 motif, LLKLL (residues 12 to 16). Our findings indicate that this conserved motif is likely to play a central role in the association between E4 and the cytokeratins. An HPV 1 E4 mutant protein containing a deletion of residues 110 to 115 induced the collapse of the cytokeratin IFs in a manner analogous to the HPV 16 E4 protein. The sequence deleted, DLDDFC, is highly conserved between cutaneous E4 proteins. HPV 1 E4 residues 42 to 80, which are rich in charged amino acids, appeared to be important in the cytoplasmic localization of E4. In addition, we have mapped the N-terminal residues of HPV 1 E4 16-kDa and 10/11-kDa polypeptides expressed by using the baculovirus system and shown that they begin at tyrosine 16 and alanine 59, respectively. Similar-sized E4 proteins are also found in vivo. N-terminal deletion proteins, which closely resemble the 16-kDa and 10/11-kDa species, expressed in keratinocytes were both cytoplasmic and nuclear but did not form cytoplasmic filamentous networks. These findings support the postulate that N-terminal proteolytic processing of the E1-- E4 protein may modulate its function in vivo.     

Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns

A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20.     

Deletion mutagenesis of the amino-terminal head domain of vimentin reveals dispensability of large internal regions for intermediate filament assembly and stability

Previous studies have shown that the non-alpha-helical head domain of vimentin is required for polymerization of intermediate filaments (IFs) and, furthermore, a nonapeptide highly conserved among type III IF subunit proteins at their extreme amino-terminus is essential for this process. Recombinant DNA technology was employed to produce specific vimentin deletion mutant proteins (for in vitro studies) or vimentin protein expression plasmids (for in vivo studies), which were used to identify other regions of the vimentin head domain important for polymerization. Various vimentin proteins lacking either residues 25-38, 44-95, or 40-95 polymerized into wild-type or largely normal IFs, both in vitro and in vivo. Vimentin proteins lacking residues 44-69 or 25-63 failed to form IFs in vitro, but assembled into IFs in vivo. Vimentin proteins lacking residues 25-68, 44-103, or 88-103 failed to form IFs in vitro or in vivo. Taken together with previous results, these data demonstrate that the middle of the vimentin non-alpha-helical head domain, which is known to be the site of nucleic acid binding, is completely dispensable for IF formation, whereas both ends of the vimentin non-alpha-helical head domain are required for IF formation. The simplest explanation for these results is that the middle of the vimentin non-alpha-helical head domain loops out, thereby permitting the juxtaposition of the ends of the head domain and their productive interaction with other protein domains (probably the C-terminus of the rod domain) during IF polymerization. The ability of some of the mutant proteins to form IFs in vivo, but not in vitro, suggests that as-yet-unknown cellular proteins may interact with and, in some cases, enable polymerization of IFs, even though they are not absolutely required for IF formation by wild-type vimentin.     

Transient coexpression of desmin and cytokeratins 8 and 18 in developing myocardial cells of some vertebrate species

During myogenesis the intermediate-sized filament (IF) cytoskeleton is characterized by increasing proportions of desmin. While skeletal and smooth muscle formation occurs in free mesenchymal cells containing vimentin-type IFs, myocardial development starts from a polar epithelium containing cytokeratin IFs and desmosomes. Therefore, we have studied the formation of the epicardium and the myocardium in different vertebrate species, combining light and electron microscopic immunolocalization techniques with gel-electrophoretic analyses of cytoskeletal proteins of microdissected myocardial tissue at differing developmental stages. In this report, we describe results obtained from advanced stages of myocardial differentiation. In all species studied the myocardial cell possess IFs abundant in desmin, often together with smaller amounts of vimentin, and the mesothelial layer of the epicardium contains cytokeratin IFs. However, we have observed remarkable interspecies differences with respect to the occurrence of cytokeratins in embryonic myocardial cells. In fetal human myocardium, from week 10 of pregnancy on, but not in juvenile and adult myocardium, and in chicken myocardium of all stages examined (until several days after hatching) specific immunostaining was seen with certain broad-range cytokeratin antibodies as well as with antibodies specific for cytokeratins 18 (in both species) and 8 (showing significant reaction only in human). This cytokeratin immunoreaction, however, did not appear in IFs extending throughout the cytoplasm or at Z-lines, but was localized in punctate arrays representing aggregates of dense material. The aggregates were often enriched at, but not restricted to, the desmosomal plaques of the intercalated discs. These observations were supported by gel-electrophoretic demonstration of small but significant amounts of cytokeratins 18 (in both species) and 8 (detected only in human) in microdissected myocardial tissue. We also observed cytokeratins in smooth muscle cells of some cardiac blood vessels. In contrast, bovine myocardium of advanced fetal age as well as rat and mouse myocardium (from fetal day 12 on) were negative for cytokeratins with all methods, although epicardial cytokeratin IFs were demonstrable. These observations are discussed in relation to myocardial histogenesis and to general problems of cytokeratin gene expression control in epithelial and nonepithelial cells.