Erika Schmid

Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells from diverse vertebrates.

Abstract Specific antibodies against vimentin, the major constitutive protein of intermediate-sized filaments present in cytoskeletons of mesenchymal cells of vertebrates, have been raised in guinea pigs. Antibodies to murine and human vimentin are of three types. The first two types produced against murine vimentin show an exclusive or preferential reaction with vimentin filaments of rodents. The third type raised against murine or human vimentin reacts with intermediate-sized filaments in species as diverse as mammals, birds and amphibia. This latter type is used here to show, both by immunoreplica techniques and by immunofluorescence microscopy, that almost all vertebrate cells growing in culture contain filaments of the vimentin type which are usually present in extended arrays. These immunological findings also suggest that the vimentin molecule contains both sequences conserved during evolution and regions different in different vertebrate species. The cells studied include not only cells of mesenchymal origin, but also cells derived from epithelia, in which it is now possible to demonstrate extensive arrays of vimentin filaments in interphase cells as well as intermediate-sized filaments of the prekeratin type. The data are consistent with the idea that most cells grown in culture contain intermediate-sized filaments of the vimentin type, irrespective of the state of differentiation of the cells from which they are derived.

Diversity of cytokeratins: Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues☆

Abstract Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [32P]phosphate labeling in vivo. It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.

Formation of Cytoskeletal Elements During Mouse Embryogenesis: Intermediate Filaments of the Cytokeratin Type and Desmosomes in Preimplantation Embryos

Different tissues of vertebrates may contain various types of intermediate-sized (7–11 nm) filaments, but little is known about their structural and functional properties during early mammalian development. We have studied the time and mode of formation of cytoskeletal elements in relation to cells and tissue differentiation during mouse embryogenesis. Here we describe the first appearance and formation of intermediate filament proteins and structures in preimplantation embryos using: (1) electron microscopy; (2) immunofluorescence microscopy with antibodies to the different types of intermediate filament proteins; and (3) two-dimensional gel electrophoresis of embryonic proteins and high salt-resistant cytoskeletal preparations. Neither intermediate filament structures nor known intermediate filament proteins have been detected between the two-cell and early-morula stage. In late morulae and blastocysts, however, three major insoluble cytoskeletal proteins (molecular weights: 46,000; 54,000; 61,000) are produced and tentatively identified as prekeratinlike proteins. Two other important cytoskeletal proteins, desmin and vimentin, are not detected. In the outer cells of morulae and in the trophectoderm of early blastocysts we have observed special junctional complexes interpreted as ‘nascent’ desmosomes because of their small size, their incomplete desmosomal plaques and midline structures, and their short tufts of associated intermediate (tonofilamentlike) filaments. In the trophectodermal cells, the number of typical (‘mature’) desmosomes and the length of bundles of densely fasciated tonofilaments extending throughout the cytoplasm increases during blastocyst growth (days 4 and 5). The prekeratinlike nature of constituent proteins of these intermediate-sized filaments has been demonstrated by their specific decoration with antibodies to bovine epidermal prekeratin and their insensitivity to colcemid treatment. Conversely, in cells of the inner cell mass of blastocysts, neither desmosomes nor intermediate-sized filaments have been observed. In the outgrowths of trophectodermal cells of blastocysts allowed to attach to and develop on cover slips for 48 h we have noticed a remarkable increase in the number of desmosomes and intermediate filaments, most of which are arranged into bundles of variable thickness. Such filament bundles are strongly stained with antibodies to prekeratin and, upon treatment of the cells with colcemid, are not aggregated into perinuclear whorls. Our results show that: 1. The first intermediate filaments formed during mouse embryogenesis are cytokeratin-type filaments present in the trophectodermal cells of blastocysts. 2. Their formation is closely associated in time and topography with the appearance of desmosomal structures. 3. We conclude from the absence of desmin and vimentin that intermediate filaments of both these types are not essential for the development of the preimplantation embryo. We further suggest that the trophectoderm resembles a differentiated cytokeratin-rich epithelium, and that the desmosome-tonofilament complex is involved in epithelial differentiation during early murine embryogenesis.

Antibody to prekeratin. Decoration of tonofilament-like arrays in various cells of epithelial character

Abstract Certain epithelial cells in culture such as the established rat kangaroo cell lines PtK1 and PtK2, mammary gland epithelial cells from cow udder, and human kidney epithelial cells are characterized by a system of wavy, branching and aggregating arrays of filaments of diameters 6–11 nm which are often desmosome-associated. Antibodies raised in guinea pigs against purified bovine prekeratin specifically decorate this system of tonofilament bundles in indirect immunofluorescence microscopy. In agreement with these results we show that preparations of these filaments isolated from such epithelial cells contain some proteins similar in polypeptide size and behaviour to components of bovine hoof prekeratin and to bovine muzzle tonofilaments. We therefore conclude that several epithelial cells which are capable of continuous division in culture continuously produce large, balanced amounts of prekeratin-like material which is assembled in tonofilament-like structures.

Biochemical and immunological identification of cytokeratin proteins present in hepatocytes of mammalian liver tissue

Abstract Hepatocytes of mammalian liver are known to contain intermediate-sized filaments of tonofilament morphology. Unlike many other epithelial cells, including cultured hepatocytes and hepatoma cells, hepatocytes present in normal liver tissue have been reported not to react, in significant intensity, with various preparations of antibodies to human and bovine epidermal prekeratin [2,6]. We have therefore examined, by biochemical and immunological methods, the cytoskeletal composition of hepatocytes grown in the body. Cytoskeletal preparations from hepatocytes of mouse and rat liver tissue resistant to high salt buffer and Triton X-100 are enriched in tangles of intermediate filaments and contain, besides some residual microfilamentous actin, a characteristic set of polypeptides. One- and two-dimensional gel electrophoresis reveals the presence of two major cytokeratin components, which appear as ‘pairs’ of isoelectric variants (component A, Mr 55 000, apparent pI values, 6.40 and 6.45; component D, Mr 49000, apparent pI values 5.43 and 5.38), and five minor components (Mr range from 41000 to 53 000), most of them also as ‘pairs’ of polypeptides slightly different in isoelectric pH value. These polypeptide patterns are very similar in mouse and rat liver although some minor but significant differences have been noted between the two species. The polypeptide patterns of liver cytoskeletons are also similar to—but clearly not identical with—the cytoskeletal protein patterns observed in other epithelial tissues and cells, including various lines of cultured rat hepatocytes and hepatoma cells. Guinea pig antibodies raised against individual cytokeratin proteins of mouse liver and against certain prekeratin polypeptides present in desmosome-attached tonofilaments of bovine muzzle are described which differ from previously described prekeratin antibodies. These prekeratin antibodies not only react with filament bundles of the prekeratin type present in many cultured epithelial cells (e.g. murine HEL, human HeLa, rat kangaroo PtK2) and various epithelial tissues, but also allow the detection of the cytokeratin components present in parenchymal cells of liver and pancreas of various species, man included. Immunofluorescence microscopy on frozen sections of liver using these antibodies reveals a novel structure, i.e. a three-dimensional filament meshwork extending throughout the whole cytoplasm of the hepatocyte, with higher intensity of staining in pericanalicular regions. The results show that parenchymal cells of normal liver and pancreas contain filaments of the cytokeratin type that are related to but not identical with epidermal prekeratin. The hepatocyte filaments appear to be different from prekeratin-type filaments present in epidermis and several other epithelial cells, both in some antigenic determinants exposed and in polypeptide composition. Our findings support the concept of the existence of a family of intermediate filament proteins, cytokeratins, containing many different polypeptides that are expressed in different epithelial cells in certain characteristic subsets in a cell type-specific mode.

Functional analysis of alternatively spliced tyrosinase gene transcripts.

Three different cDNA clones (pmcTyr1, pmcTyr2 and pmcTyr3) representing mRNAs originating by alternative splicing of the primary transcript of mouse tyrosinase gene, were identified and characterized by sequence analysis and by a functional assay. These cDNAs were subcloned into the newly constructed expression vector pHD. After electroporation of these hybrid clones into tyrosinase negative cells, protein extracts were prepared and tested for tyrosinase enzyme activity. Only the cDNA insert of pmcTyr1 was able to confer tyrosinase enzyme activity. This cDNA encodes a protein 533 amino acid residues in length containing a putative leader peptide of 18 amino acids and six putative glycosylation sites. Comparisons of the deduced amino acid sequence of the cDNA clone pmcTyr1 with the protein sequence of tyrosinases from man, Streptomyces, Neurospora and with haemocyanin subunits from a spider showed two regions of sequence conservation. One of these regions is known to be involved in copper binding. Since this gene with the coding capacity for tyrosinase is absent in all studied c‐locus lethal deletion mutant mice, we have evidence that albinism in mice is caused by mutations of the tyrosinase gene.

Intermediate filament proteins in nonfilamentous structures: Transient disintegration and inclusion of subunit proteins in granular aggregates

The intermediate filament cytoskeleton of cultured bovine kidney epithelial cells and human HeLa cells changes dramatically during mitosis. The bundles of cytokeratin and vimentin filaments progressively unravel into protofilament-like threads of 2-4 nm diameter, and intermediate filament protein is included in numerous, variously sized (0.2-1.5 microns) spheroidal aggregates containing densely stained granular particles of 5-16 nm diameter. We describe these mitotic bodies in intact cells and in isolated cytoskeletons. In metaphase to anaphase of normal mitosis and after colcemid arrest of mitotic stages, many cells contain all their detectable cytokeratin and vimentin material in the form of such spheroidal aggregate bodies, whereas in other mitotic cells such bodies occur simultaneously with bundles of residual intermediate filaments. In telophase, the extended normal arrays of intermediate filament bundles are gradually reestablished. We find that vimentin and cytokeratins can be organized in structures other than intermediate filaments. Thus, at least during mitosis of some cell types, factors occur that promote unraveling of intermediate filaments into protofilament-like threads and organization of intermediate filament proteins into distinct granules that form large aggregate bodies. Some cells, at least certain epithelial and carcinoma cells, may contain factors effective in structural modulation and reorganization of intermediate filaments.

Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one‐ or two‐dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the ‘basic cytokeratin subfamily’ defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis‐specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad‐range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.

HeLa cells contain intermediate-sized filaments of the prekeratin type

Abstract Immunofluorescence microscopy using antibodies raised against protein constituents of the different types of intermediate-sized filaments has shown that in HeLa cells filaments containing a prekeratin-like protein (cytokeratin) predominate. The wavy filament bundles decorated by antibodies against prekeratin are similar to those described in other cells of epithelial origin. These bundles of intermediate-sized (6–11 nm) filaments are also described by electron microscopy in intact cells and in cytoskeletal preparations obtained by cell lysis and extraction with low and high salt buffers and Triton X-100. The occasional occurrence of desmosome-attached tonofibrillar bundles of intermediate-sized filaments is also shown. When HeLa cells are treated for long times with colcemid to induce perinuclear whorls of intermediate-sized filaments, these aggregates of filaments are strongly stained by antibody to vimentin, the major polypeptide of the intermediate-sized filaments of murine 3T3 cells. However, they are not stained by antibody to prekeratin, and the display of the prekeratin-containing tonofilament-like structures is similar to that seen in untreated cells. SDS-polyacrylamide gel electrophoresis of cytoskeletons prepared under conditions in which intermediate-sized filaments are retained show the presence of a polypeptide which co-migrates with one component of bovine prekeratin, and a second polypeptide which co-migrates with vimentin purified from mouse 3T3 cells. The data show ( i ) that two different types of intermediate-sized filaments can be present in the same cell and can be distinguished immunologically; and ( ii ) that the expression of a prominent epithelial structural marker, i.e. prekeratin-containing filaments, can be maintained in malignancy and continuing proliferation in vitro.

Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta

Abstract The presence of intermediate filament proteins in vascular tissue cells has been examined by immunofluorescence microscopy on frozen sections of the aortic wall of diverse vertebrates (rat, cow, human and chicken) and by gel electrophoresis of cytoskeletal proteins from whole aortic tissue or from stripped tunica media of cow and man. Most cells of the aortic wall in these species contain vimentin filaments, including smoooth muscle cells of the tunica media. In addition, we have observed aortic cells that are positively stained by antibodies to desmin. The presence of desmin in aortic tissue has also been demonstrated by gel electrophoresis for rat, cow and chicken. In aortic tissue some smooth muscle cells contain both types of intermediate filament proteins, vimentin and desmin. Bovine aorta contains, besides cells in which vimentin and desmin seem to co-exist, distinct bundles of smooth muscle cells, located in outer regions of the tunica media, which contain only desmin. The results suggest that (i) intermediate-sized filaments of both kinds, desmin and vimentin, can occur in vascular smooth muscle in situ and (ii) smooth muscle cells of the vascular system are heterogeneous and can be distinguished by their intermediate filament proteins. The finding of different vascular smooth muscle cells is discussed in relation to development and differentiation of the vascular system.