Immunolocalization of intermediate filament proteins using antibodies to synthetic peptides.

Synthetic peptides corresponding to amino acid sequences of amino terminal non-alpha helical domains of human cytokeratin 18 and to low molecular weight human neurofilament subunit were used to obtain monospecific antisera. The results of our immunohistochemical investigations confirmed in general the data previously published on the distribution of cytokeratin 18 in human, rat, and calf tissues. The reactivity of the antiserum was abolished after formalin fixation of specimens. Immunolocalization of the neurofilament subunit using our monospecific antiserum was quite variable from species to species in cells of the central and peripheral nervous systems, and also varied as the result of the tissue fixation procedures. In particular, formalin fixation destroyed the immunoreactivity of the recognized epitope. We discuss the advantages and limits of the use of synthetic peptides as immunogens to produce polyclonal antibodies against intermediate filament proteins, with particular attention to the epitope masking phenomena in cytokeratin polypeptides and the phosphorylation of epitopes in neurofilament subunits.     

Characteristics of specific intermediate filament proteins in human brain tumors

The content of glial fibrillary acidic protein (GFAP) was measured in human brain tumours with different histological structure, origin and rate of malignancy. The polypeptide composition of CFAP was established in human brain and tumours by SDS polyacrylamide gel electrophoresis followed by immunoblotting. In tumours with an astrocyte type of differentiation, GFAP was revealed as a set of immunologically related and partially degraded polypeptides with a molecular weight of around and below 37 kD. It was assumed that the appearance of intact GFAP polypeptides (49 kD) in some tumours may be considered as a result of penetration of reactive astrocytes into tumour tissue.     

Immunomorphologic study of vertebrate renal glomerula using antibodies to intermediate filament proteins

A comparative immunomorphologic study was carried out on cryostate sections of renal tissue of plaice (Pleuronectes platessa L.) and rat, using specific antibodies against the proteins of intermediate filaments--cytokeratins and vimentin. No cytokeratins were revealed in cells of renal glomerula in both the animals under investigation. Indirect immunofluorescence of the polyclonal serum against vimentin showed brightly coloured capillaries of the renal glomerula of plaice and weakly coloured ones of rat. At the same time cells of parietal epithelium of the Bowman capsule showed trace or negative reaction. The electron microscopic control revealed a powerful development of intermediate filament system in the podocyte cytoplasm of plaice, and a dense microfilament network and plural bundles of microtubules in the podocyte cytoplasm of rats. Problems of conservatism of the vimentin intermediate filaments in the evolution are discussed in addition to the present theories of the origin and development of renal glomerula of the vertebrates.     

Characterisation and tissue-specific expression of the two keratin subfamilies of intermediate filament proteins in the cephalochordate Branchiostoma

The cloning of three intermediate filament proteins expressed at the gastrula stage (kl, Y1, X1) extends the size of the IF multigene family of Branchiostoma to at least 13 members. This is one of the largest protein families established for the lancelet. Sequence comparisons indicate five keratin orthologs, three of type I (E1, k1, Y1) and two of type II (E2, D1). This assignment is confirmed by the obligatory heteropolymeric polymerisation behaviour of the recombinant proteins. In line with the hetero-coiled-coil principle IF are formed by any stoichiometric mixture of type I and II keratin orthologs. In spite of the strong sequence drift chimeric IF are formed between K8, a human keratin II, and two of the lancelet type I keratins. We discuss whether the remaining 8 IF proteins reflect three additional and potentially cephalochordate-specific subfamilies. The tissue-specific expression patterns of the 5 keratins and some other IF proteins were analysed by immunofluorescence in the adult. Keratins are primarily present in ectodermally derived tissues. Developmental control of the expression of some IF proteins is observed, but three keratins (k1, Y1, D1) and an additional IF protein (X1) detected at the gastrula stage are expressed throughout the life cycle.     

The use of antibodies to intermediate filament proteins in the differential diagnosis of lymphoma versus metastatic carcinoma

Summary Forty-nine cases encompassing 16 different types of malignant lymphoma were examined for their intermediate filament protein (IFP) type by indirect immunofluorescence microscopy of cryostat sections. In all cases, vimentin was shown to be the only IFP type detectable in these tumours. Lymphomas are negative for keratin and desmin, which are characteristic for benign and malignant epithelial or muscular tissues respectively. In addition, eighteen cases are described in which antibodies to intermediate filament proteins were used successfully to distinguish between lymphoma and metastatic carcinoma where differential diagnosis was difficult or impossible on the basis of routine histology.     

Flow-cytometric analysis of mixed cell populations using intermediate filament antibodies

Using two human tumour cell lines, T24 bladder carcinoma and Molt-4 leukemia, flow-cytometric DNA analysis of pure and mixed cell populations was performed using cellular cytokeratin content to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratin. Using cytokeratin content to gate DNA analysis, the same specificity and sensitivity of cellular DNA content and distribution measurement could be achieved by single-pass FCM analysis of a mixture of the two cell types as was seen when analysing pure populations of the two cell lines. This technique has broad applicability to FCM analysis of mixed populations composed of cells from different tissues of origin.     

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.     

Association of syncoilin and desmin: linking intermediate filament proteins to the dystrophin-associated protein complex.

We recently identified a novel protein called syncoilin, a putative intermediate filament protein that interacts with alpha-dystrobrevin, a member of the dystrophin-associated protein complex. Syncoilin is found at the neuromuscular junction, sarcolemma, and Z-lines and is thought to be important for muscle fiber integrity. Based on the similar protein structure and cellular localization of syncoilin and desmin, we proposed that these proteins interact in vivo. The data presented confirm an interaction between syncoilin and desmin and demonstrate their co-localization in skeletal muscle. Intriguingly, whereas these proteins interact, COS-7 cell expression studies show that desmin and syncoilin do not assemble into heterofilaments. Furthermore, fractionation assay and immunofluorescence study of H2K myoblasts and myotubes suggest that, unlike typical intermediate filament proteins, syncoilin does not participate in filament formation with any protein. However, it is possible that syncoilin is involved in the anchoring of the desmin intermediate filament network at the sarcolemma and the neuromuscular junction. This interaction is likely to be important for maintaining muscle fiber integrity and may also link the dystrophin-associated protein complex to the cytoskeleton. The dysfunction or absence of syncoilin may result in the disruption of the intermediate filament network leading to muscle necrosis. Syncoilin is therefore an ideal candidate gene for muscular dystrophies and desmin-related myopathies.     

Immunomorphological study of 1,2-dimethylhydrazine-induced carcinomas of the large intestine in rats using monoclonal antibodies to intermediate filament proteins

Cryostatic sections of rat large bowel tumors induced by 1,2-dimethylhydrazine were stained with monoclonal antibodies against different proteins of intermediate filaments: (a) against prekeratin (mol. mass 49 000, PK49) found in many epithelial cells and (b) against vimentin, a constituent of intermediate filaments of mesenchymal cells. Immunofluorescence study showed that large bowel tumor cells as well as normal cells of this organ contain PK49 but not vimentin. High sensitivity of the method allowed one to clearly identify small invasive nodules and groups of tumor cells not visible in usual histologic preparations. Moreover, in some cases single atypical tumor cells were identified in tumor stroma and in the submucosal layer underlying the tumor, that were indistinguishable from normal mesenchymal cells at the light microscopy level.     

Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells

Monoclonal antibodies were prepared against a 46,000 mol wt major cytoplasmic protein from Drosophila melanogaster Kc cells. These antibodies reacted with the 46,000 and a 40,000 mol wt protein from Kc cells. Some antibodies showed cross-reaction with 55,000 (vimentin) and 52,000 mol wt (desmin) proteins from baby hamster kidney (BHK) cells that form intermediate sized filaments in vertebrate cells. In indirect immunofluorescence, the group of cross reacting antibodies stained a filamentous meshwork in the cytoplasm of vertebrate cells. In Kc cells the fluorescence seemed to be localized in a filamentous meshwork that became more obvious after the cells had flattened out on a surface. These cytoskeletal structures are heat-labile; the proteins in Kc or BHK cells rearrange after a brief heat shock, forming juxtanuclear cap structures.     

Polyclonal antibodies to chlorosome proteins as probes for green sulfur bacteria.

We found that polyclonal antibodies raised against chlorosome polypeptides from green sulfur bacteria reacted to Chlorobium tepidum, Chlorobium limicola, and Chlorobium phaeobacteroides but not to Chloroflexus aurantiacus. These antibodies successfully labeled only green sulfur species in marine microbial mat samples. Our results suggest that these antibodies may be useful as immunohistochemical probes.     

An immunohistochemical study of early embryogenesis in the clawed toad Xenopus laevis by using monoclonal antibodies to intermediate filament proteins

Distribution of cytokeratin epitopes was studied in X. laevis embryos at stages 10-25 using 5 monoclonal antibodies against proteins of the human and rat keratin filaments. Specific staining was observed in chorda, outer layers of ectoderm and presumptive epidermis (late gastrula), and inner layer of presumptive epidermis. The cells of the stained zone (presumptive epidermis) were compressed while the cells of unstained zone (presumptive neuroectoderm) were extended tangentially.     

Immunofluorescent quantification of tyrosine phosphorylated proteins by flow cytometric analysis

To measure the quantity of tyrosine phosphorylated proteins in cells, we have used a flow cytometry technique with fluorescein isothiocyanate-labeled anti-phosphotyrosine antibody (FITC-PY mAb). The analysis was applied to the phosphotyrosine titration and showed an optimum amount of FITC-PY mAb (30g/1×10cells). The staining specificity of our assay was tested by the addition of exogenous competitors, showing a specific inhibition by phosphotyrosine but not by phosphoserine, phosphothreonine, or tyrosine. The assay was also able to elucidate the inhibitory effect on tyrosine phosphorylation of genistein, a protein tyrosine kinase inhibitor. These results imply that immunofluorescent quantification assay using a flow cytometer could be a useful technique to determine the intracellular level of tyrosine phosphorylated protein.     

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor.     

Periodic charge distribution in the intermediate filament proteins desmin and vimentin

A helical coiled-coil region of amino acid sequence surrounding the cysteine residue of desmin and vimentin shows a regular pattern of alternating positive and negative charges with periods close to $\text{28}\text{3}$ and $\text{28}\text{10}$ residues. This suggests relationships with the charge distributions of myosin rod and alpha-keratin. The common features may reflect a similar pattern of three-dimensional packing in vivo for each of these molecules.     

Antibodies to intermediate filament proteins in the immunohistochemical identification of human tumours: an overview

Intermediate-sized filament proteins (IFP) are tissue specific in that antibodies to keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and the neurofilament proteins can distinguish between cells of epithelial and mesenchymal origin as well as of myogenic and neural origin respectively. Malignant cells retain their tissue-specific IFP, which makes it possible to use these antibodies in tumour diagnosis. Carcinomas are exclusively detected by antibodies to keratin. Monoclonal antibodies to keratin have allowed the differentiation between subgroups of epithelial tumours until now between adenocarcinomas and squamous cell carcinomas. Lymphomas, melanomas and several soft tissue tumours are distinctly recognized by antibodies to vimentin. On the other hand, rhabdomyosarcomas and leiomyosarcomas are positive for desmin, while astrocytomas give a strong reaction with GFAP antibodies. Thus, antibodies to IFP are useful tools for differential diagnosis in surgical pathology.     

Developmentally controlled expression patterns of intermediate filament proteins in the cephalochordate Branchiostoma

Expression of cytoplasmic intermediate filament (IF) proteins starts in the gastrula with three keratins (k1, Y1, D1) and protein X1. The number of IF proteins expressed increases at the neurula and early larval stages to seven and 11, respectively, and reaches 13 in the adult. Using antibodies specific for a single IF protein the expression patterns of nine of the 13 IF proteins were analyzed at different developmental stages. Keratin k1 of the larval epidermis is replaced in the juvenile by keratin E1. Protein C1 of the larval epidermis persists only weakly and only in the most ventral part of the adult. While down-regulated in the adult epidermis k1 and C1 are major proteins in the atrial epithelium which forms in the later larva. B1 is currently the only IF protein expressed in mesodermally derived tissues such as the muscle tails and some coelomic epithelia. Two-dimensional gels confirm that keratins are the major IF proteins in the nerve cord. Immunogold electronmicroscopy shows that proteins X1 and C2 are present in epidermis and nerve cord in keratin IF.     

Rapid detection of phosphotyrosine proteins by flow cytometric analysis in Bcr-Abl-positive cells.

BACKGROUND: Constitutive tyrosine phosphorylation derived from Bcr-Abl kinase activity is the major characteristic of Bcr-Abl positive cells. In this study, we developed a method to detect the phosphotyrosine proteins by flow cytometry and we asked whether phosphorylation was affected by imatinib mesylate treatment. METHODS: Cells were treated or not with imatinib mesylate, fixed and permeabilized by PFA followed by saponin, then stained with anti-phosphotyrosine (p-tyr) monoclonal antibody and analyzed by flow cytometry. RESULTS: Optimal staining parameters were performed with p-tyr antibody using K562 and LAMA84 lines that displayed high levels of tyrosine phosphorylation as compared to the control line, HL60. Tyrosine phosphorylation was inhibited by imatinib in a dose-dependent manner, but not modified by other inhibitors demonstrating that the staining detected is specific to Bcr-Abl phosphorylation. The staining of imatinib-resistant cell lines such as the mutated BaF/Bcr-AblT315I cell line or resistant CML patient cells, showed that hyperphosphorylation was not affected by imatinib treatment. In one CML patient, our technique permitted us to detect a small hyperphosphorylated population resistant to imatinib that appeared hyperphosphorylated and amplified at the time of relapse. CONCLUSIONS: We have developed a flow cytometric technique presenting several advantages such as rapidity and sensitivity, which requires fewer cells than the Western blot.