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.     

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.     

Relationship between tektins and intermediate filament proteins: an immunological study

Affinity-purified antibodies raised against three flagellar tektins (tektin A, B, and C) from each of two sea urchin species (Lytechinus pictus and Strongylocentrotus purpuratus) were used to study the immunological relationship between tektins and intermediate filament proteins. By immunofluorescence microscopy, several antitektins revealed a staining of intermediate filament-like arrays in three vertebrate cell lines tested. Immunoelectron microscopy substantiated the cross reaction of antitektins with intermediate filaments. When the cells were treated with cytochalasin B, the arrangement of the filaments recognized by anti-(Lp)-tektin B was altered; the alteration observed is typical for keratin filaments. By immunoblot, it was found that anti-(Lp)-tektin B cross reacted with two isoforms or different proteins of approximately 54 kD with pIs of 6.1 and 6.2 in human carcinoma epithelia (HeLa) cells and with two isoforms or different proteins of approximately 55 kD with pIs of 6.1 and 6.3 in pig kidney epithelia (LLC-PK1) cells. Furthermore, when antitektin antibodies were affinity purified with the 54 kD HeLa keratin, these keratin-specific antibodies again restained the original tektins on immunoblots. From these observations, it can be concluded that tektins and keratins are to a certain extent immunologically related. To determine the degree of the immunological relationship, tektin filaments and purified intermediate filaments from HeLa cells were cleaved with alpha-chymotrypsin and examined by quantitative immunoblot analysis. On immunoblots of digested tektins from L. pictus, anti-(Lp)-tektin B recognized several cleavage products in the range of 20 kD to 46 kD. However, when immunoblots of digested intermediate filaments from HeLa cells were probed, the cross reaction of anti-(Lp)-tektin B with HeLa keratins was eliminated by more than 98% within 2 min, suggesting that tektins have epitopes in common with the end domains of certain keratins.     

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.     

Occurrence of two different intermediate filament proteins in the same filament in situ within a human glioma cell line : An immunoelectron microscopical study

Intermediate filament systems of an established glioma cell line have been characterized by double immunofluorescence microscopy and by immunoelectron microscopy using two antibodies, one of which recognizes glial fibrillary acid protein (GFA) but not vimentin, and the second which recognizes vimentin but not GFA. The results show that glioma cells express two immunologically distinct IF polypeptides which are found in the same 10-nm filaments. Juxtanuclear caps formed after exposure of the cells to colcemid consisted of intermediate filaments composed of both GFA and vimentin. In immunoelectron microscopy both untreated cells and cells treated with colcemid show discontinuous labelling when only a single antibody is used, but continuous labelling when both antibodies are used simultaneously.     

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.     

The beaded filament of the eye lens: an unexpected key to intermediate filament structure and function

In 1959, an unusual filamentous polymer, now called the beaded filament, was described in the lens of the eye. The constituent proteins, assembly properties and functions of the beaded filament have been elusive. The recent publication of the sequences for two major lens filament proteins (CP49 and filensin) and the reconstitution in vitro of structures closely resembling beaded filaments, suggests that the beaded filament is related structurally to intermediate filaments (IFs). The association of the lenticular chaperones, the alpha-crystallins, with the filament contributes to the characteristic beaded morphology, as well as giving important clues to the function of this unusual filament in the lens. These recent results have several implications for IF function and assembly.     

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.     

Cytoplasmic intermediate filament protein expression in tunicate development: a specific marker for the test cells

The urochordate Ciona intestinalis is a well established system for embryological studies, and large scale EST sequences begin to emerge. We cloned five cytoplasmic intennediate filament (IF) cDNAs and made specific antibodies to the recombinant proteins. Self-assembly studies and immunofluorescence microscopy were used to study these proteins and their distribution. Confirming and extending previous studies in Styela, we found that Ciona protein IF-A is expressed in muscle and forms homopolymeric filaments while proteins IF-C and IF-D, which form only obligatory heteropolymeric filaments, resemble a keratin pair exclusively found in the entire epidermis. Protein IF-B and the new protein IF-F potentially reflect tunicate-specific IF proteins. They are found in the entire internal epithelia including the neural gland. We also extended the analysis to earlier developmental stages of Ciona. Protein IF-A is expressed in muscle from larval stages, whereas proteins IF-C and IF-D are found only in the tail epidermis. Protein IF-F is detected abundantly in the test cells of eggs, embryos and premetamorphic larvae. Our studies show that IF proteins could prove very useful markers in the study of cell fate determination in Ciona. They also support previous findings on the evolutionary relationships of different IF proteins. Non-vertebrate chordates have IF proteins which represent orthologs of vertebrate type I to III proteins, but also IF proteins that do not seem to fit into these classes. However, the intron positions of all tunicate IF genes are conserved with vertebrate type I to III genes, pointing to a common evolutionary origin.     

The intermediate filament complement of the retina: a comparison between different mammalian species

We compared the intermediate filament expression of the various cell types in the fully differentiated neural retina from rat, mouse, rabbit, guinea pig, cow, pig, and cat. Many cell types had an intermediate filament complement conserved across species boundaries, such as Müller cells and retinal ganglion cells. In some species (rabbit, guinea pig, and cow), however, we were unable to visualize GFA (glial fibrillary acidic)-positive retinal astrocytes, although such profiles were clearly visible in the remainder. Horizontal cell staining proved to be extremely species-variable. In rat and mouse the processes of these cells were identically displayed with antibodies to vimentin and all three neurofilament triplet proteins. In cow they decorated with antibodies to vimentin and antibodies to the two lower molecular weight neurofilament proteins alone, whereas in pig, rabbit and guinea pig all three neurofilament proteins but not vimentin were present. Finally cat horizontal cells stained for all three neurofilament proteins, some finer processes being additionally stainable with vimentin. A further surprise was the visualization of profiles positive only for the two lower molecular weight neurofilament proteins in the inner nuclear layer of both rabbit and guinea pig retina but not the other species. The implications of these results will be discussed.     

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.     

An immunofluorescence microscopical study of the neurofilament triplet proteins, vimentin and glial fibrillary acidic protein within the adult rat brain

A collection of antibodies specific to different intermediate filament proteins were applied to frozen sections of adult rat brains. The relative distribution of these proteins was then studied using double label immunofluorescence microscopy. Antibodies specific to each of the neurofilament "triplet" proteins (of approximate molecular weight 68 K, 145 K and 200 K) stained exclusively neuronal structures. The distribution of these three antigens was in general identical, except that certain neurofilament populations such as those in the dendrites and cell bodies of pyramidal cells of the hippocampus and cerebral cortex, contained relatively little if any 200 K protein. Some neurone populations, such as the granule cells of the cerebellar cortex, could not be visualized by neurofilament antibodies, indicating that neurofilaments may not be essential for function of all neurones in vitro. Antibodies to GFA and vimentin stained an entirely different population of processes, none of which stained with any of the neurofilament antibodies. Vimentin antibody stained sheath material around the brain, a monolayer of ependymal cell bodies lining the ventricles, fibrous material associated within the choroid plexus, the walls of blood vessels and capillaries, and the processes of cells in certain regions. GFA antibody stained a second layer of sheath material under the vimentin layer, and numerous processes visible throughout the brain. Some specific populations of GFA-positive processes proved to stain also with vimentin. These included the processes of Golgi "epithelial" cells (Bergmann glial fibres), those of certain astrocytes in bundles of myelinated fibers. In addition, some processes apparently derived from ependymal cells proved to stain for both vimentin and GFA, whilst other could only be reliably visualized by vimentin alone. These results are discussed in terms of the previously described morphological characteristics of the various cell types of the brain.     

Production of specific antibodies to contractile proteins and their use in immunofluorescence microscopy

Summary The preparation of highly purified myosin from surgical specimen of human uterine muscle is described. Antibodies were raised in rabbits against this immunogen. In immunodiffusion, they react with uterine and chicken gizzard muscle myosin, no reaction is observed between uterine myosin and the anti-chicken-gizzard- myosin. In immunofluorescence, antiuterine-myosin stains smooth muscle in the contractile and modulated state and non-muscle cells such as fibroblasts, platelets and endothelium of various species. Thus, these antibodies contrast anti-gizzard-myosin, which has previously been shown to be specific for contractile state muscle cells. We therefore conclude that the uterine myosin preparation consists of two immunogens, the one being associated with cell contractility and the other, termed cytoplasmic myosin, with motility and mitosis. The latter is indistinguishable from the myosin present in non-muscle cells and can be absorbed specifically with actomyosin from blood platelets.Abbreviations ATP Adenosine triphosphate - DNAse I Deoxyribonuclease I - DTE Dithioerythritol - SDS Sodiumdodecylsulfate - PAGE Polyacrylamide electrophoresis     

Proteolysis of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins.

The degradation of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins proceeds in two stages in the form of a limited proteolysis. At first, the reaction is very rapid, with the stepwise and complete removal of a peptide (ca. 9,000 daltons) from the N-terminal of vimentin and desmin. This results in the production of a characteristic "staircase" of degradation products, as seen in two-dimensional polyacrylamide gel electrophoresis. The second stage of proteolysis is characterized by the accumulation of peptides which are resistant to further proteolysis; this is due not to product inhibition but to the fact that these peptides are not substrates for the proteinase and therefore do not protect the latter from inactivation (autodigestion). In vitro phosphorylation of the substrates does not affect proteinase activity, probably because the phosphorylation site is located towards the C-terminal of the molecules. The specific and limited proteolysis of vimentin and desmin results in the deletion of the nucleic acid binding and filament assembly site of these proteins, indicating that the Ca2+-activated proteinase plays a role in regulating the function(s) of these intermediate filament proteins, rather than their simple turnover during the cell cycle.     

Production of specific antibodies to contractile proteins,and their use in immunofluorescence microscopy

Summary The injection of rabbits with insoluble or soluble G-actin from chicken smooth or striated muscle will produce antibodies that are equally reactive, and species and tissue non-specific in immunoprecipitation, immunofluorescence and actin-activated Mg²⁺-ATPase^** inhibition test. These antibodies have been used for the identification of actin-containing fibrils in a variety of tissues. When G-actins from chicken smooth or striated muscle are immobilized by chemical linkage to Affi-Gel 702 microbeads, their immunogenicity is increased, but the antibodies obtained against them are species-specific and will only react with actin and actin-containing structures from chicken and are therefore limited in use. It is concluded from this work that insoluble G-actin is the preferable immunogen to obtain precipitating antibodies for wide use.Abbreviations ATPase Adenosinetriphosphatase - FITC Fluoresceinisothiocyanate - SDS Sodiumdodecylsulfate This paper is dedicated to Dr. Dorothy M. Needham, University of Cambridge, England, in honour of her eightieth birthday     

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.     

Production of specific antibodies to contractile proteins,and their use in immunofluorescence microscopy

Summary Antibodies prepared against actomyosins can be shown to behave similarly, if not identically to more recently prepared antibodies against highly purified myosins. Details of the purification of the antigens, and of the production of antibodies to chick myosins from smooth gizzard muscle and from striated pectoral muscle are given. The antibody specificity appears to be directed against the heavy chains of the myosin molecules, since these antibodies specifically inhibit the myosin ATPase reaction, and sincein situ staining of myosin polypeptide chains on an SDS gel using the antibodies in indirect fluorescence shows staining only in the heavy band region. Use of the antibodies in immunofluorescence microscopy suggest that the antibodies are tissue, but not species, specific. Example of their use in staining tissue sections are shown.     

XY body formation during rat spermatogenesis: an immunocytochemical study using antibodies against XY body-associated proteins

The process of formation of the XY body during meiotic prophase was investigated by immunocytochemistry on cryosections of pubertal rat testes using antibodies against three different XY body-associated proteins. Here we show that these proteins are detectable at only partially overlapping temporal windows. These findings provide the first evidence that the previously described morphological changes in the structure of the XY body that occur during meiotic prophase are accompanied by considerable changes in its protein composition. Received: 22 May 1997 / Accepted: 14 June 1997