Intermediate filaments in muscle and epithelial cells of nematodes.

Current concepts of the developmentally controlled multigene family of intermediate filament (IF) proteins expect the origin of their complexity in evolutionary precursors preceding all vertebrate classes. Among invertebrates, however, firm ultrastructural as well as molecular documentation of IFs is restricted to some giant axons and to epithelia of a few molluscs and annelids. As Ascaris lumbricoides is easily dissected into clean tissues, IF expression in this large nematode was analyzed by electron microscopic and biochemical procedures and a monoclonal antibody reacting with all mammalian IF proteins. We document for the first time the presence of IFs in muscle cells of an invertebrate. They occur in three muscle types (irregular striated pharynx muscle, obliquely striated body muscle, uterus smooth muscle). IFs are also found in the epithelia studied (syncytial epidermis, intestine, ovary, testis). Immunoblots on muscles, pharynx, intestine, uterus, and epidermis identify a pair of polypeptides (with apparent molecular masses of 71 and 63 kD) as IF constituents. In vitro reconstitution of filaments was obtained with the proteins purified from body muscle. In the small nematode Caenorhabditis elegans IF proteins are so far found only in the massive desmosome-anchored tonofilament bundles which traverse a special epithelial cell type, the marginal cells of the pharynx. We speculate that IFs may occur in most but perhaps not all invertebrates and that they may not occur in all cells in large amounts. As electron micrographs of the epidermis of a planarian--a member of the Platyhelminthes--reveal IFs, the evolutionary origin of this cytoplasmic structure can be expected either among the lowest metazoa or already in some unicellular eukaryotes.

Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits.

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.

Modulation of desmin intermediate filament assembly by a monoclonal antibody.

We have used a monoclonal antibody against desmin to examine the assembly of intermediate filaments (IF) from their building blocks, the tetrameric protofilaments. The antibody, designated D76, does not cross react with any other IF proteins (Danto, S.I., and D.A. Fischman. 1984. J. Cell Biol. 98:2179-2191). It binds to a region amino-terminal to cys-324 of avian desmin that is resistant to chymotrypsin and trypsin digestion, and in the electron microscope appears to bind to the ends of tetrameric protofilaments. In combination, these findings suggest that the epitope of the antibody resides at the amino-terminal end of the alpha-helical rod domain. Preincubation of desmin protofilaments with an excess of D76 antibodies blocks their subsequent assembly into IF. In the presence of sub-stoichiometric amounts of antibodies, IF are assembled from protofilaments but they are morphologically aberrant in that (a) they are capped by IgG molecules at one or both ends; (b) they are unraveled to varying degree, revealing a characteristic right-handed helical arrangement of sub-filamentous strands of different diameters. The antibody binds only to the ends but not along the length of desmin IF. The most straightforward explanation for this is that the epitope resides in a part of the desmin molecule that becomes buried within the core of the filament upon polymerization and is therefore inaccessible to the antibody.

Organization of desmosomal plaque proteins in cells growing at low calcium concentrations.

Desmosomes are not formed in epithelial cell cultures growing in media with low (less than or equal to 0.1 mM) concentrations of Ca2+ (LCM) but appear rapidly upon shift to media of normal calcium concentrations (NCM). Previous authors using immunolocalization of desmoplakin, a marker protein for the desmosomal plaque, in LCM-grown cells have interpreted positively stained, dense, cytoplasmic aggregates on intermediate filaments (IF) bundles as preformed plaque units which upon NCM shift would move to the plasma membrane and contribute to desmosome formation. Studying various cell cultures, including primary mouse keratinocytes and human A-431 cells, we show that most, probably all, desmoplakin-positive aggregates in LCM-grown cells are associated with membranous structures, mostly vesicles, and also contain other desmosomal markers, including desmoglein, a transmembrane glycoprotein. We interpret such vesicles as residual desmosome-derived domains endocytosed upon cell dissociation. Only keratinocytes grown for long times (2-4 wk) in LCM are practically free from such vesicles. In addition, we demonstrate that certain cells such as A-431 cells, when passaged in LCM and in the absence of stable junctions, are able to continually assemble "half-desmosomes" on the plasma membrane which in turn can be endocytosed as plaque-bearing vesicles. We also show that in LCM the synthesis of several desmosomal proteins (desmoplakins I and II, plakoglobin, desmoglein, "band 6 protein") continues and that most of the plaque protein, desmoplakin, is diffusely spread over the cytoplasm, apparently in a soluble monodisperse form of approximately 9S. From our results we propose that the plaque proteins occur in small, discrete, diffusible entities in the cytoplasm, in concentrations that are relatively high in LCM and low in NCM, from which they assemble directly, i.e., without intermediate precursor aggregates on IFs in the cytoplasm, on certain plasma membrane domains in a Ca2+ dependent process.

Isoprenylation is required for the processing of the lamin A precursor.

The nuclear lamina proteins, prelamin A, lamin B, and a 70-kD lamina-associated protein, are posttranslationally modified by a metabolite derived from mevalonate. This modification can be inhibited by treatment with (3-R,S)-3-fluoromevalonate, demonstrating that it is isoprenoid in nature. We have examined the association between isoprenoid metabolism and processing of the lamin A precursor in human and hamster cells. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin (lovastatin) specifically depletes endogenous isoprenoid pools and inhibits the conversion of prelamin A to lamin A. Prelamin A processing is also blocked by mevalonate starvation of Mev-1, a CHO cell line auxotrophic for mevalonate. Moreover, inhibition of prelamin A processing by mevinolin treatment is rapidly reversed by the addition of exogenous mevalonate. Processing of prelamin A is, therefore, dependent on isoprenoid metabolism. Analysis of the conversion of prelamin A to lamin A by two independent methods, immunoprecipitation and two-dimensional nonequilibrium pH gel electrophoresis, demonstrates that a precursor-product relationship exists between prelamin A and lamin A. Analysis of R,S-[5-3H(N)]mevalonate-labeled cells shows that the rate of turnover of the isoprenoid group from prelamin A is comparable to the rate of conversion of prelamin A to lamin A. These results suggest that during the proteolytic maturation of prelamin A, the isoprenylated moiety is lost. A significant difference between prelamin A processing, and that of p21ras and the B-type lamins that undergo isoprenylation-dependent proteolytic maturation, is that the mature form of lamin A is no longer isoprenylated.

The cDNA-deduced amino acid sequence for trichohyalin, a differentiation marker in the hair follicle, contains a 23 amino acid repeat.

Trichohyalin is a highly expressed protein within the inner root sheath of hair follicles and is similar, or identical, to a protein present in the hair medulla. In situ hybridization studies have shown that trichohyalin is a very early differentiation marker in both tissues and that in each case the trichohyalin mRNA is expressed from the same single copy gene. A partial cDNA clone for sheep trichohyalin has been isolated and represents approximately 40% of the full-length trichohyalin mRNA. The carboxy-terminal 458 amino acids of trichohyalin are encoded, and the first 429 amino acids consist of full- or partial-length tandem repeats of a 23 amino acid sequence. These repeats are characterized by a high proportion of charged amino acids. Secondary structure analyses predict that the majority of the encoded protein could form alpha-helical structures that might form filamentous aggregates of intermediate filament dimensions, even though the heptad motif obligatory for the intermediate filament structure itself is absent. The alternative structural role of trichohyalin could be as an intermediate filament-associated protein, as proposed from other evidence.

Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins.

The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deep-etched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's cross-linking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solid-phase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.

Expression of epidermal keratins and filaggrin during human fetal skin development.

The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a novel 66 kd subunit of mammalian neurofilaments.

A 66 kd protein, pl 5.4, was purified from the Triton-insoluble fraction of rat spinal cord. This protein formed 10 nm filaments in vitro. The 66 kd protein was unique, although it shared homology with the 70 kd neurofilament protein (NF-L) and vimentin. An antiserum (anti-66) specific to the 66 kd protein did not cross-react with any of the neurofilament triplet proteins. In the spinal cord, anti-66 intensely stained the axons of the anterior and lateral columns. However, afferents from dorsal root ganglia and the efferents from the motoneurons were negative. In the cerebellum, anti-66 intensely stained most axons. The 66 kd protein was readily detectable in homogenates of forebrain, cerebellum, brainstem, and spinal cord, but was found only in trace amounts in adult sciatic nerves and was not found in extraneural tissues. The 66 kd protein constituted 0.5% of total protein in the spinal cord, whereas NF-L constituted about 1.5%.

Chemically induced tumors of rat olfactory epithelium: a model for human esthesioneuroepithelioma.

N-Nitrosopiperidine (CAS: 100-75-4) and 2,6-dimethylnitrosomorpholine induced tumors of the olfactory epithelium in white Wistar rats. Some tumors were serially transplanted to NMRI nude mice (nu/nu) and passaged up to 16 times in a 1-year period. Tumor tissues from rats and mice were analyzed by conventional pathological stains, by electron microscopy, and by immunofluorescence microscopy with the use of antibodies specific for different intermediate filaments. Both carcinogens induced tumors built of undifferentiated small, round cells in which neuroblastic (Homer-Wright) rosettes and ependymal (Flexner) rosettes were visible. In some tumors areas of squamous cell metaplasia could be observed, which sometimes differentiated toward squamous cell carcinoma. Electron microscopy showed neurosecretory granules in some tumor cells, and biochemical studies of plasma showed in some instances elevated ACTH and calcitonin levels. Intermediate filament typing showed that in general the undifferentiated tumor cells lack intermediate filaments, although in 6 of 29 tumors a few cells that stained positively for neurofilaments were found. Flexner rosettes, the areas showing squamous cell differentiation, and occasional single tumor cells were positive with keratin antibodies. Neurofilament expression was observed in a minor population of tumor cells placed in tissue culture. These findings are used to argue that the chemically induced rat tumors are a model for human esthesioneuroepithelioma and furthermore that the light basal cells of the epithelium may be the stem cells of the rat tumors as well as of its rare counterparts in humans.

Immunocytochemical detection of human lung cancer heterogeneity using antibodies to epithelial, neuronal, and neuroendocrine antigens.

Lung cancers were investigated for their heterogeneity as expressed by their immunoreactivity for cytokeratins and neurofilament proteins, as well as for the neuroendocrine differentiation antigen MOC-1. Using broadly cross-reacting antibodies, cytokeratins were detected in nearly all cases of lung carcinomas. Keratinization could be detected only in cases of moderately to well-differentiated squamous cell carcinoma (SQC) using a monoclonal antibody to cytokeratin 10, while a monoclonal antibody reactive with cytokeratin 18, and specific for glandular epithelia, reacted with adenocarcinomas, small cell lung carcinomas (SCLC), and lung carcinoids. In SQC this antibody could detect non-squamous cell differentiation, showing increasing numbers of positive cells with decrease of histologically detectable SQC differentiation. Cells positive for neurofilaments were demonstrated in some of the poorly differentiated SQCs and in some of the cases of SCLC, possibly representing the variant type of SCLC. Also in some of the lung carcinoids neurofilament proteins were present, colocalizing with cytokeratins. MOC-1 was present in all SCLC and lung carcinoids. This antibody could also detect neuroendocrine differentiation in all combined small cell carcinomas, in one poorly differentiated adenocarcinoma, and in about 30% of the poorly differentiated SQCs. Therefore, lung cancer heterogeneity can be detected using a panel of well-defined antibodies to intermediate filaments in combination with the MOC-1 antibody. The use of these antibodies in diagnosis can have prognostic significance and can lead to a more selective therapeutic approach.

Induction of cells with phenotypic features of neuronal cells by treatment with dibutyryl cyclic adenosine 3',5'-monophosphate in a human parotid gland adenocarcinoma cell line in culture.

A human parotid gland adenocarcinoma cell line, with an intercalated duct cell phenotype of the salivary gland and expression of vasoactive intestinal polypeptide and amylase, was cultivated in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP). Morphological changes occurred; cells formed long cytoplasmic processes densely packed with ample microfibrils, as well as microtubules, and grew in a netlike appearance. In addition, it has been found by the immunofluorescence staining technique, immunoblotting, or immunoelectron microscopy that the cells treated with dB-cAMP express neurofilaments, neuron-specific enolase, synaptophysin, and HNK-1 antigen, as well as the alpha- and beta-chains of tubulin, whereas these antigens are not detected in untreated cells. The expression of vasoactive intestinal polypeptide detected diffusely in the cytoplasm of untreated cells was restricted to the cell membranes during the cultivation of cells in the presence of dB-cAMP, while expression of amylase persisted in the treated cells in a fashion similar to that in untreated cells. Moreover, both anchorage-independent and anchorage-dependent growth of the cells was markedly suppressed in the presence of dB-cAMP. After removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype and growth rate of the untreated cells. These findings indicate that reversible conversion into cells with phenotypic features of neuronal cells of a human parotid adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Cytokeratin filament organization in the ciliated cells of the quail oviduct.

With the exception of keratinocytes and some types of cultured cells, ciliated cells appear to be the major cell type which contains the most developed cytokeratin meshwork. We report, here, on the intermediate filament (IF) organization in ciliated cells of the quail oviduct using ultrastructural and immunocytochemical techniques. Special attention was focused on the relationships between IF and other cell organelles. The meshwork of IFs appears as a subapical disk constituted of separate bundles mainly composed of interwoven 10-nm filaments. From this subapical region, a descending bundle connects the array of IFs occupying the basal part of the cell. The nucleus is maintained in a loose network of IFs. In ciliated cells there are no free centrioles, but IFs are related to centriolar appendages (striated rootlets).

Filamentous cross-bridges link intermediate filaments to the nuclear pore complexes.

Intermediate filament-nuclear matrix interactions were studied in cultured rat ventral prostate cells and isolated rat uterine epithelial cells. Cytokeratin filaments were identified by immunoelectron microscopy. In addition to conventional thin section of Triton X-100 treated cells, subcellular residues composed of intermediate filaments and nuclear matrix were critical-point dried and platinum-carbon replicated. The results demonstrate the presence of a previously unrecognized type of filamentous cross-bridges that link intermediate filaments to the nuclear pore complexes.

Expression of rat neurofilament proteins NF-L and NF-M in transfected non-neuronal cells.

Two cDNA clones fully encoding the rat neurofilament proteins NF-L and NF-M were subcloned into eukaryotic expression vectors behind the strong constitutive viral promoters from SV40 and Rous sarcoma viruses. Transient transfection of L tk- and Cos cell lines with these expression constructs resulted in cells expressing the neurofilament proteins in an intermediate filament-type pattern. Additionally, a putative juxtanuclear organizing center or region was observed in the transfected cells, most noticeable shortly after the transfection procedure. Stable transfections were performed on mouse L tk- and Swiss 3T6 cells using NF-L and NF-M constructs bearing an SV40 early promoter driven neomycin selectable marker. Although G418-resistant clones were recovered with both the NF-L and the NF-M constructs, only clones expressing immunofluorescently stainable amounts of NF-M were detected and established. Immunoelectron microscopic analysis revealed NF-M and vimentin proteins to be colocalized on the same intermediate filaments.

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis.

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.

Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments.

Monoclonal antibodies specific for vimentin (V9), keratin 7 (CK 7) and keratin 18 (CK5) have been microinjected into three human epithelial cell lines: HeLa, MCF-7 and RT-4. The effect of the injection on other keratin polypeptides and vimentin filaments has been observed by double label immunofluorescence and in some instances by immunoelectron microscopy using gold labels of different sizes. Microinjection of V9 into HeLa cells causes the vimentin to collapse into a perinuclear cap leaving the keratin filaments unaffected. Injection of CK5 does not affect the vimentin filaments but disrupts the keratin filaments revealing keratin aggregates similar to those seen in some epithelial cell lines during mitosis. The keratin aggregates obtained after microinjection in HeLa contain the keratins 8 and 18 and probably also other keratins, as no residual keratin filaments are observed with a keratin polyclonal antibody of broad specificity. Aggregates in mitotic HeLa cells contain at least the keratins 7, 8, and 18. In MCF-7 cells keratins 8, 18, and 19 are observed in the aggregates seen 3 h after microinjection which, however, show a different morphology from those seen in HeLa cells. In MCF-7 cells a new keratin filament is built within 6 h after the injection which is composed mainly of keratin 8 and 19. The antibody-complexed keratin 18 remains in spherical aggregates of different size. The results suggest that in HeLa cells vimentin and keratin form independent networks, and that individual 10 nm filaments in epithelial cell lines can contain more than two keratins.

Mitochondrial binding of a protein affected in mutants resistant to the microtubule inhibitor podophyllotoxin.

Specific antibodies to a protein P1 Mr approximately equal to 63,000) from Chinese hamster ovary cells, which is affected in mutants resistant to the microtubule inhibitor, podophyllotoxin, and behaves like a microtubule-related protein by certain criteria [14], have been raised. The antibody reacts specifically with the P1 protein in one- and two-dimensional immunoblots, and a cross-reacting protein of similar molecular mass and electrophoretic mobility is also found in cells from various vertebrate and invertebrate species. The observed similarity in the peptide maps of the cross-reacting protein from human, mouse, Chinese hamster and chicken cells indicates that the structure of this protein should be highly conserved. However, no P1-antibody cross-reacting protein was observed in plants (corn, mung), fungus (Neurospora crassa), yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli and Salmonella typhimurium). Immunofluorescence studies with the P1-antibody show that, in interphase cells of various cross-reacting species, it bound specifically to mitochondria which were associated and distributed on and along the length of microtubules. Similar association and codistribution of mitochondria and microtubules were not observed in mitotic cells. Some implications of the mitochondrial localization of the protein P1 and the observed association between microtubules and mitochondria are discussed.

Effects of pemphigus antibody on the regeneration of cell-cell contact in keratinocyte cultures grown in low to normal Ca++ concentration.

Pemphigus is an autoimmune blistering disease of epidermal cells in which autoantibodies to the surface develop. The present study was performed to determine whether the binding of pemphigus antibodies to the surface of keratinocytes can inhibit the regeneration of cell-cell contact induced by altering from low to normal Ca++ concentration medium. Human keratinocytes (a cell line of squamous cell carcinoma, DJM-1 cell) were grown in low Ca++ medium for 4 days, then the cells were incubated in normal Ca++ medium containing 10% pemphigus (4 patients with pemphigus vulgaris and 4 patients with pemphigus foliaceus) or normal serum (treated at 56 degrees C, for 30 min) for various incubation periods (2, 6, 12, 24 h). The cells were fixed and stained with antikeratin antibody by the indirect immunofluorescence method so that the detachment of cell-cell contact was able to be clearly visualized by observing the cytoskeletal arrays of keratin filaments. The cells grown in normal Ca++ medium showed detachments of cell-cell contact 24-36 h after addition of any one of the pemphigus sera used in this study. The cells grown in low Ca++ medium formed no cell-cell contacts and expressed no pemphigus antigens. However, re-formation of cell-cell contacts and reexpression of the antigens were confirmed by immunofluorescence microscopy 30 min after the addition of Ca++ to the medium. The addition of any pemphigus vulgaris and foliaceus sera with Ca++ did not inhibit the regeneration of cell-cell contact and exerted no effects on the contact during the subsequent 12 h. However, after 24 h, these cells again lost the contact. These results indicate that pemphigus antibody and antigen reaction on the cell surface did not directly inhibit the Ca++-induced re-formation of cell-cell contact.

A 48 kilodalton intermediate filament associated protein (IFAP) in reactive-like astrocytes induced by dibutyryl cyclic AMP in culture and in reactive astrocytes in situ.

In this paper we demonstrate that the 48 kilodalton (kDa) intermediate filament associated protein (IFAP), previously reported to be present in normal astrocytes, is also present in reactive astrocytes in situ and in reactive-like astrocytes induced by dibutyryl cyclic AMP in vitro. This IFAP is detectable by antibodies in normal rabbit serum (F2N) and is closely associated with glial fibrillary acidic protein-containing intermediate filaments (IF). The expression of 48 kDa IFAP is related to the acquisition of stellate shape by normal and reactive-like astrocytes in vitro. It is proposed that 48 kDa IFAP may be responsible for cross-linking IF into bundles and is thereby associated with cell process formation.