Mary Osborn

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.

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.

Chapter 7 Immunofluorescence and Immunocytochemical Procedures with Affinity Purified Antibodies: Tubulin-Containing Structures

Publisher Summary This chapter describes the study of the intracellular location of proteins by immunocytochemical procedures. Although the methods given in the chapter are generally applicable, they are documented for tubulin containing structures and antibodies to tubulin. Further examples of the usefulness of these methods in studying cell structure can be found in several recent reviews. Immunofluorescence microscopy is a very sensitive method, theoretically requiring only one antigenic site on the structural protein. It offers several unique advantages for the study of cytoskeletal structures. First, only the protein of choice is visualized by use of its specific antibody. Second, if the protein forms part of a supramolecular structure, the organization of that structure throughout the cell can be demonstrated. Third, numerous cells can be observed simultaneously, and thus the unique features of the organization under different experimental conditions can be observed. Thus, immunofluorescence microscopy can bridge the gap between light microscopy and electron microscopy.

The detergent-resistant cytoskeleton of tissue culture cells includes the nucleus and the microfilament bundles

Abstract Mouse 3T3 and chick embryo cells grown in monolayers have been treated with the non-ionic detergents, NP40 or Triton X-100, to give “nuclear monolayers”. Immunofluorescence microscopy with antibodies against actin shows that most of the microfilament bundles remain detergent resistant and form part of the cells cytoskeleton. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis of cytoskeleton preparations from chick embryo fibroblasts show the following major proteins: the lower molecular weight histones, a protein coelectrophoresing with actin and a protein, X, of molecular weight approx. 58 000 which is different from tubulin. Thus, at least in well spread cells containing a strongly developed system of microfilamentous bundles, the detergent-resistant cytoskeleton includes the nucleus, large amounts of the 58 000 molecular weight protein and the microfilamentous bundles. The importance of the existence of microfilamentous actin in the cytoskeleton is discussed in relation to previous reports on the existence of actin as a major non-histone protein in the nucleus.

An F-actin- and calmodulin-binding protein from isolated intestinal brush borders has a morphology related to spectrin

A high molecular weight protein from the brush border of chicken intestinal epithelial cells has been purified. This protein (TW 260/240), a complex of two polypeptides with apparent molecular weights of 260,000 and 240,000, accounts for a significant amount of the terminal web organization. TW 260/240 is an F-actin-binding protein that also interacts with calmodulin. Rotary shadowing reveals long flexible rods of double-stranded morphology tightly connected at each end. TW 260/240 is quite distinct from smooth muscle filamin and macrophage actin-binding protein (APB), but, in spite of its higher contour length (265 nm), seems to be related to erythrocyte spectrin (194 nm for the tetramer). Immunofluorescence microscopy with antibodies against TW 260/240 indicates the existence of a submembranous organization distinctly different from that of stress fibers. We have compared TW 260/240 with fodrin, a brain protein known to occur in submembranous organization but not previously characterized in molecular terms. TW 260/240 and fodrin are clearly distinct molecules but are similar in many aspects. Ultrastructural, biochemical and immunological results indicate three distinct classes of rod-like high molecular weight actin-binding proteins, possibly reflected by the prototypes filamin (ABP), spectrin and TW 260/240 (fodrin). The latter group may be responsible for calmodulin control of submembranous microfilament structures in various nonmuscle cells.

Monoclonal antibodies to desmin, the muscle-specific intermediate filament protein.

A set of monoclonal antibodies to desmin has been isolated from a fusion of mouse myeloma cells with spleen cells from mice immunized with purified porcine desmin. Eleven group I antibodies recognized desmin in the immune blot, and using defined desmin fragments the epitope has been tentatively assigned as lying between residues 325 and 372. When cell lines were tested in immunofluorescence only the human line RD and hamster BHK‐21 were positive. When tissue sections were used, skeletal, cardiac, visceral and some vascular smooth muscle cells were positive. Thus, the group I antibodies appear specific for desmin and do not recognize other intermediate filament proteins. Group II monoclonals recognized not only desmin in the immune blot but also other polypeptides. The epitope of this class is located between residues 70 and 280. In immunofluorescence on cell lines and tissues, the staining patterns of group II antibodies were more complicated and demonstrate that not only other intermediate filament proteins but also additional antigenic determinants are being recognized. The group I antibodies stain, as expected from their desmin specificity, rat and human rhabdomyosarcomas and thus appear to be useful reagents in pathology.

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.

Intermediate-sized filament proteins (prekeratin, vimentin, desmin) in the normal parotid gland and parotid gland tumours

Antibodies to different intermediate-sized filament proteins can distinguish cells and tissues of epithelial, mesenchymal, muscle, astrocytic and neural origin. Antibodies to prekeratin, vimentin and desmin have been used to distinguish cells of epithelial, mesenchymal and muscle origin in the normal human parotid gland, and in addition to study some common tumors of this gland. Prekeratin-positive and vimentin-positive cells are found among the tumor cells in the pleomorphic adenomas. In contrast the tumor cells of the mucoepidermoid tumors and squamous cell carcinomas are prekeratin-positive but vimentin-negative.

Monoclonal cytokeratin antibodies that distinguish simple from stratified squamous epithelia: characterization on human tissues.

Four monoclonal antibodies designated CK1 ‐ CK4 were obtained from fusions of mouse myeloma F0 cells with spleen cells from BALB/c mice immunized with cytoskeletal preparations made by treatment of human HeLa cells with non‐ionic detergents. These IgG1 type antibodies all recognize, in immune blots, cytokeratin 18 (45 kd, pI 5.7) in the catalogue of 19 human cytokeratin species developed by Moll et al. (1982). Immunofluorescence microscopy on human material shows that CK1 ‐ CK4 stain a wide variety of simple epithelia (e.g., intestine, respiratory and urinary systems, liver, glandular epithelia) but do not stain stratified squamous epithelia (e.g., oesophagus, epidermis) or non‐epithelial cells. The immunofluorescence results, developed mainly by gel electrophoresis, support the concept of cytokeratin divergence in different epithelia and clarify, for cytokeratin 18, some unsolved problems posed by high tissue complexity. CK2 appears specific for human, CK1 and CK3 for primates, while CK4 shows broad cross‐species reactivity. Thus, CK1 ‐ CK4 appear to be valuable tools for cytokeratin typing and initial experiments also suggest that they can be used to further subdivide human tumours of epithelial origin.

Direct demonstration of the presence of two immunologically distinct intermediate-sized filament systems in the same cell by double immunofluorescence microscopy. Vimentin and cytokeratin fibers in cultured epithelial cells

Abstract The epithelial derived cell lines PtK2 and HeLa were characterized by double immunofluorescence microscopy using purified antibodies against vimentin and prekeratin. The results show that both cell types express simultaneously two immunologically distinct intermediate-sized filaments. Use of colcemid-treated cells confirms that the vimentin fibers and not the keratin-related fibers are rearranged into coils around the nucleus. In some cells staining of fibrous fragments is observed, which are perhaps involved in the synthesis or breakdown of this class of filaments. The concept that growing cells derived from differentiated cell types express not only the intermediate-sized filament system typical of the differentiated cell type but in addition contain intermediate-sized filaments of the vimentin type is discussed.