Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Heterogeneity of tubulin epitopes in mouse fetal tissues

A panel of six monoclonal antibodies against alpha (TU-01, TU-03, TU-04, TU-05, TU-09) or beta (TU-13) subunits of tubulin was used to study expression of tubulin epitopes in 14-day-old mouse embryos. Specificity of antibodies was confirmed by immunoblotting experiments. Monoclonal antibodies TU-01, TU-09 and TU-13, like the polyclonal antibody reacted essentially with all tissues, whereas other antibodies displayed differential reactivity. Most notably, TU-03 reacted very strongly with simple epithelia and basal layer of stratified epithelial layers. TU-04 recognized maturation related changes in spinal cord. Reactivity of TU-05 was restricted to central nervous system and peripheral nerves. Present results document immunohistochemical heterogeneity of tubulin in fetal tissues and suggest the existence of maturation and tissue specific epitopes of tubulin in developing organs.     

Heterogeneity of tubulin epitopes in mouse fetal tissues

Summary A panel of six monoclonal antibodies against alpha (TU-01, TU-03, TU-04, TU-05, TU-09) or beta (TU-13) subunits of tubulin was used to study expression of tubulin epitopes in 14-day-old mouse embryos. Specificity of antibodies was confirmed by immunoblotting experiments. Monoclonal antibodies TU-01, TU-09 and TU-13, like the polyclonal antibody reacted essentially with all tissues, whereas other antibodies displayed differential reactivity. Most notably, TU-03 reacted very strongly with simple epithelia and basal layer of stratified epithelial layers. TU-04 recognized maturation related changes in spinal cord. Reactivity of TU-05 was restricted to central nervous system and peripheral nerves.Present results document immunohistochemical heterogeneity of tubulin in fetal tissues and suggest the existence of maturation and tissue specific epitopes of tubulin in developing organs.     

Heterogeneity of microtubules recognized by monoclonal antibodies to alpha-tubulin

A set of four monoclonal antibodies against tubulin (TU-01, TU-02, TU-03, and TU-04) were produced using pig brain microtubule protein as antigen. Their characterization shows that all recognize antigenic determinants located on the tubulin alpha-subunit. However, peptide mapping of isolated alpha-tubulin, followed by immunoblotting with the monoclonal antibodies, shows that the antigenic determinants are located on different peptide fragments in at least three cases. The immunoreactivity with tubulins from different cells and tissues, ranging from eukaryotic microorganisms to man, was studied by immunoblotting and immunofluorescence. The antigenic determinants recognized by the antibodies are not uniformly distributed but, in some instances, are absent from tubulins of lower eukaryotic cells. These antibodies also make it possible to distinguish between different sets of microtubules within individual cells. Antigenically different microtubules are particularly evident in mouse spermatozoa and in some protozoa (T. vaginalis, H. muscarum, L. tropica, N. gruberi) possessing different sets of microtubules with different functions. These monoclonal antibodies can clearly identify the heterogeneity of tubulin or microtubules both from different organisms and within the same cell.     

Immunostaining of human spermatozoa with tubulin domain-specific monoclonal antibodies. Recognition of a unique beta-tubulin epitope in the sperm head.

Four monoclonal antibodies that discriminate between structural domains of alpha-(TU-01, TU-04) or beta-(TU-06, TU-12) tubulin and a polyclonal anti-tubulin antibody were used for immunostaining of human spermatozoa using immunofluorescence microscopy. Specificity of antibodies was confirmed by immunoblotting experiments. Antibodies TU-01 and TU-06 uniformly stained the whole tail and the neck, whereas antibodies TU-04, TU-12 showed differential distribution of corresponding epitopes in the stable arrays of flagellar microtubules. Of the monoclonal antibodies used, only TU-12 against the antigenic determinant on C-terminal domain of beta-tubulin showed strong reactivity with the equatorial segment of the head. The results document a differential exposure of tubulin epitopes at the single-cell level and suggest the existence of distinct tubulin populations in various structural compartments of the human spermatozoon.     

Immunostaining of human spermatozoa with tubulin domain-specific monoclonal antibodies. Recognition of a unique beta-tubulin epitope in the sperm head

Summary Four monoclonal antibodies that discriminate between structural domains of alpha-(TU-01, TU-04) or beta-(TU-06, TU-12) tubulin and a polyclonal anti-tubulin antibody were used for immunostaining of human spermatozoa using immunofluorescence microscopy. Specificity of antibodies was confirmed by immunoblotting experiments. Antibodies TU-01 and TU-06 uniformly stained the whole tail and the neck, whereas antibodies TU-04, TU-12 showed differential distribution of corresponding epitopes in the stable arrays of flagellar microtubules. Of the monoclonal antibodies used, only TU-12 against the antigenic determinant on C-terminal domain of -tubulin showed strong reactivity with the equatorial segment of the head. The results document a differential exposure of tubulin epitopes at the single-cell level and suggest the existence of distinct tubulin populations in various structural compartments of the human spermatozoon.     

Changing antigenic pattern of tubulin-containing structures during spreading ofMizuhopecten yessoensis (Mollusca) hemocytes

Summary The immunoreactivity of a panel of anti-tubulin monoclonal antibodies with spreadingMizuhopecten yessoensis hemocytes was studied by immunofluorescence and immunoblotting. In immunoblotting all the antibodies used reacted only with bands corresponding to the position of tubulin subunits. Hemocytes showed a reorganization of microtubules and microfilaments during cell spreading. In spread-out cells the TU-04 antibody stained microtubules growing out of the centriole in the cell body; in contrast to TU-07 and TU-10 antibodies, which stained microspike-like bundles on the periphery of the cells. The presence of microfilaments in microspikes was detected by rhodamine-labeled phalloidin.Abbreviations CB cytoskeletal buffer - SWAM-FITC fluorescein isothiocyanate-labeled swine anti mouse immunoglobulin - MTOC microtubule organizing centers - SDS-PAGE SDS polyacrylamide gel electrophoresis     

Distinct subcellular localization of β-tubulin epitopes in the adult mouse brain

 A panel of monoclonal antibodies specific of α-tubulin (TU-01, TU-09) and β-tubulin (TU-06, TU-13) subunits was used to study the location of N-terminal structural domains of tubulin in adult mouse brain. The specificity of antibodies was confirmed b immunoblotting experiments. Immunohistochemical staining of vibratome sections from cerebral cortex, cerebellum, hippocampus, and corpus callosum showed that antibodies TU-01, TU-09, and TU13 reacted with neuronal and glial cells and their processes, whereas the TU-06 antibody stained only the perikarya. Dendrites and axons were either unstained or their staining was very weak. As the TU-06 epitope is located on the N-terminal structural domain of β-tubulin, the observed staining pattern cannot be interpreted as evidence of a distinct subcellular localization of β-tubulin isotypes or known post-translational modifications. The limited distribution of the epitope could, rather, reflect differences between the conformations of tubulin molecules in microtubules of somata and neurites or, alternatively, a specific masking of the corresponding region on the N-terminal domain of β-tubulin by interacting protein(s) in dendrites and axons. Accepted: 11 November 1996     

Charge variants of tubulin,tubulin S,membrane-bound and palmitoylated tubulin from brain and pheochromocytoma cells

Isoelectric focusing (IEF) of only approximately 1 microg of rat brain tubulin yields 27-30 distinct charge variants in the pH range of 4.5-5.4 with band separations of 0.01-0.02 pH units as detected by silver staining. Variants can be efficiently transferred from the immobilized gradient strip to polyvinylidene difluoride (PVDF) membranes for reaction with monoclonal antibodies. C-terminal-directed antibodies to alpha- and beta-tubulin yield patterns similar to N-terminal-directed antibodies. Removal of the acidic C-termini with subtilisin to form tubulin S increases the pI values by approximately 1 pH unit, leads to a loss in the isoelectric distinction between the alpha- and beta-tubulin variants seen by N-terminal-directed antibodies, and abolishes reactions with all beta-variants and all but three alpha variants by C-terminal-directed antibodies (TU-04 and TU-14). Many, but not all, of the variants are substrates for autopalmitoylation of rat brain tubulin. The distribution of isoelectric variants differs between cytoplasm and membrane fractions from PC12 pheochromocytoma cells. A potential role for different variants is suggested.     

Localization and characterization of a specific linear epitope of the Brucella DnaK protein

We have previously produced and characterized four monoclonal antibodies to the Brucella DnaK protein which were derived from mice infected with B. melitensis or immunized with the B. melitensis cell wall fraction. By use of a recombinant DNA technique, we have localized a linear epitope, recognized by two of these monoclonal antibodies (V78/07B01/G11 and V78/09D04/D08), in the last 21 amino acids of the C-terminal region of the Brucella DnaK protein. The C-terminal region has been reported to be the most variable region among DnaK proteins. The two other monoclonal antibodies (A53/09G03/D02 and A53/01C10/A10) failed to react with the recombinant clones and might recognize discontinuous epitopes of the Brucella DnaK protein. The four monoclonal antibodies reacted with all recognized Brucella species and biovars in immunoblotting after SDS-PAGE. Monoclonal antibodies V78/07B01/G11 and V78/09D04/D08 did not react with reported cross-reacting bacteria nor with bacteria of the α-2 subdivision of the class Proteobacteria for which a close genetic relationship with Brucella spp. has been reported. However, monoclonal antibodies A53/09G03/D02 and A53/01C10/A10 reacted with Phyllobacterium rubiacearum and/or Ochrobactrum anthropi, both bacteria of the α-2 subdivision of the class Proteobacteria. The Brucella genus DnaK specific epitopes could be of importance for diagnostic purposes.     

The bovine desmocollin family: a new gene and expression patterns reflecting epithelial cell proliferation and differentiation

We have discovered a third bovine desmocollin gene, DSC3, and studied expression of all three desmocollin genes, DSC1, 2, and 3, by Northern blotting, RT-PCR and in situ hybridization. DSC1 is strongly expressed in epidermis and tongue papillae, showing a "skin"-type pattern resembling that previously described for keratins 1 and 10. Expression is absent from the epidermal basal layer but appears in the immediate suprabasal layers and continues uniformly to the lower granular layer. In tongue epithelium, expression is suprabasal and strictly localized to papillae, being absent from interpapillary regions. In other epithelial low level DSC1 expression is detectable only by RT-PCR. The distribution of Dsc1 glycoproteins, detected by an isoform-specific monoclonal antibody, closely reflects mRNA distribution in epidermis and tongue. DSC2 is ubiquitously expressed in epithelia and cardiac muscle. In stratified epithelia, expression appears immediately suprabasal, continuing weakly to the lower granular layer in epidermis and to just above half epithelial thickness in interpapillary tongue, oesophageal, and rumenal epithelia. DSC3 expression is restricted to the basal and immediately suprabasal layers in stratified epithelia. In deep rete ridges DSC expression strikingly resembles the distribution of stem, transit-amplifying, and terminally differentiating cells described by others. DSC3 expression is strongly basal, DSC2 is strong in 5-10 suprabasal layers, and then weakens to be superseded by strong DSC1. These results suggest that desmocollin isoform expression has important functional consequences in epithelial proliferation, stratification, and differentiation. The data also provide a standard for nomenclature of the desmocollins.     

A broad spectrum monoclonal antibody to alpha-tubulin does not recognize all protozoan tubulins

Summary Monoclonal antibodies able to recognize single antigenic determinants are a powerful tool for the study of immunological heterogeneity of antigens. In this paper we have used a monoclonal antibody against the -subunit of pig brain tubulin (TU-01) to investigate the immunoreactivity of tubulins from mammals, avians, amphibia, echinodermata, plathelmints, slime moulds and protozoa. Immunoreactivity was detected using immunoblotting and indirect immunofluorescence of isolated cells. Our results show that the antigenic determinant recognized by the TU-01 antibody is present in all metazoan tubulin tested and among the eukaryotic microorganisms only in the flagellateTrichomonas vaginalis. Indirect immunofluorescence also reveals that not allTrichomonas microtubules are stained by TU-01 antibody indicating the presence of different tubulins within a single cell. This results are consistent with the multitubulin hypothesis (Fulton andSimpson 1976).     

Heterogeneity of keratin distribution in the oral mucosa and skin of mammals as determined using monoclonal antibodies

Summary The immunohistochemical localization of keratins in the oral epithelia of several mammals was investigated using the monoclonal antibodies to keratins, PKK1 (41–56 kilodaltons) and KL1 (55–57 kilodaltons). The staining patterns obtained in different locations of the oral mucosa and of the skin epidermis were compared. In the papillae on the dorsal surface of the tongue, some areas exhibited marked PKK1 staining, while other area were PKK1 negative. In general, rodent oral epithelia were negative for PKK1 in the basal layer, while comparatively strong PKK1 staining was observed in cells of the upper spinous layer. In the epidermis, positive PKK1 reactions were confined to the basal layer, while KL1 staining was occasionally seen in the basal layer of oral epithelia. In cats, dogs, and monkeys, different PKK1 and KL1 binding patterns were observed in oral epithelia. Also, the distribution in oral epithelia differed from that seen in the epidermis of these animals. In the epidermis, the distribution of PKK1 and KL1 was regular, with PKK1 usually being confined to the basal layer, while KL1 binding was found in the spinous and granular cell layers, and was dependent on the degree of keratinization. In the animals studies, keratin expression as detected by PKK1 and KL1-was different in the skin epidermis and oral epithelia, and the localization of these keratins differed in the various types of oral mucosa.     

Heterogeneity of keratin distribution in the oral mucosa and skin of mammals as determined using monoclonal antibodies

The immunohistochemical localization of keratins in the oral epithelia of several mammals was investigated using the monoclonal antibodies to keratins, PKK1 (41-56 kilodaltons) and KL1 (55-57 kilodaltons). The staining patterns obtained in different locations of the oral mucosa and of the skin epidermis were compared. In the papillae on the dorsal surface of the tongue, some areas exhibited marked PKK1 staining, while other area were PKK1 negative. In general, rodent oral epithelia were negative for PKK1 in the basal layer, while comparatively strong PKK1 staining was observed in cells of the upper spinous layer. In the epidermis, positive PKK1 reactions were confined to the basal layer, while KL1 staining was occasionally seen in the basal layer of oral epithelia. In cats, dogs, and monkeys, different PKK1 and KL1 binding patterns were observed in oral epithelia. Also, the distribution in oral epithelia differed from that seen in the epidermis of these animals. In the epidermis, the distribution of PKK1 and KL1 was regular, with PKK1 usually being confined to the basal layer, while KL1 binding was found in the spinous and granular cell layers, and was dependent on the degree of keratinization. In the animals studies, keratin expression--as detected by PKK1 and KL1--was different in the skin epidermis and oral epithelia, and the localization of these keratins differed in the various types of oral mucosa.     

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Received: 2 May 1996 / Accepted: 16 September 1996     

Comparative histochemical study of Bowen's disease and actinic keratosis: preserved normal basal cells in Bowen's disease

The degree of DNA-instability as revealed by immunohistochemical staining with anti-cytidine antibody after acid hydrolysis (DNA-instability test) has been recently used as a marker of malignancy. This technique was applied to examine 17 skin tissue samples of Bowen's disease, 47 of actinic keratosis, 15 of squamous cell carcinoma, 5 of seborrheic keratosis, and 10 of normal skin. All benign neoplastic cells of seborrheic keratosis and normal epidermal cells were negative. On the other hand, all cancer cells were positive with the DNA-instability test, indicating their malignancy, but all basal cells in Bowen's disease were completely negative. Compatible with this result, the basal cells in Bowen's disease were characteristically normal as evident in other histochemical examinations. Thus, they were negative with p53 immunohistochemistry, with normal signals of chromosome 17 in situ hybridisation and argyrophilic nucleolar organiser region, and showed slightly enhanced proliferative activity as revealed by proliferating cell nuclear antigen immunohistochemistry. Immunohistochemical staining with 34 beta E12 (monoclonal antibody against cytokeratins 1, 5, 10, and 14), which stains all normal epidermal keratinocytes including basal cells, showed that only the basal cells of Bowen's disease stained strongly and homogeneously, while all cancer cells in the upper layers of Bowen's disease and all layers of actinic keratosis were only sporadically or weakly stained. Staining with 34 beta B4 (monoclonal antibody against cytokeratin 1), which recognises the whole epidermis except for the basal layer in the normal epidermis, showed that the basal cells in the Bowen's disease were completely negative, and lower layer cells in the actinic keratosis and upper layer cells in Bowen's disease were only sporadically stained positive, although the superficial layer cells in actinic keratosis stained strongly and homogeneously. Our findings clearly indicate that the basal cells in Bowen's disease are normal. In support of this conclusion, the same cells showed normal morphology on electron microscopy with preserved basement membrane, although the latter was often damaged in actinic keratosis.     

Distinct antigenic characteristics of murine parietal yolk sac laminin

Two monoclonal antibodies (LAM-A and LAM-B) specific for laminin from normal and neoplastic parietal yolk sac (PYS) cells were produced in rats immunized with a mouse yolk sac carcinoma cell line. Both antibodies immunoprecipitated the 400,000- and 200,000-Da chains of laminin and reacted with purified PYS laminin in ELISA. LAM-A reacted with mouse and rat PYS laminin, whereas LAM-B reacted only with mouse PYS laminin. Formaldehyde- and methanol-fixed adult and fetal somatic tissues were immunohistochemically unreactive with either of the two antibodies. In acetone-fixed tissue sections, both antibodies reacted weakly with some medullary tubules of the kidney, the follicular basement membrane of the ovaries, and the seminiferous tubules. The antibodies appear to react with the polypeptide determinants residing on the terminal portion of the long arm of laminin. It is concluded that laminin derived from normal or malignant PYS cells has distinct antigenic sites that are immunochemically not apparent in most other basement membranes.     

Localisation of P2X5 and P2X7 receptors by immunohistochemistry in rat stratified squamous epithelia

In order to investigate whether purinoceptors are involved in the physiological renewal and regeneration of epithelia, we used immunohistochemical techniques on fresh frozen sections of various stratified squamous epithelial tissues (cornea, tongue, soft palate, oesophagus, vagina and footpad) of the rat and specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. Only two of the antibodies, anti-P2X5 and anti-P2X7, reacted with epithelial structures. P2X5 immunoreactivity was mainly associated with the membranes of the proliferating and differentiating cell layers (spinous and granular layer) in both keratinised and non-keratinised epithelia and growing hair follicles. In contrast, P2X7 immunoreactivity was clearly associated with the keratinisation process, the staining being most intense in the upper keratinised and the exfoliated layers. These findings suggest, for the first time, that P2X5 and P2X7 receptors play an important role in the physiological turnover of continuously regenerating cells, and further, raise the possibility that they represent novel targets for the development of pharmacological tools of potential benefit for diseases of epithelial dysfunction.     

Differential localization of G proteins, Gi and Go, in the olfactory epithelium and the main olfactory bulb of the rat.

The immunohistochemical localization of proteins Gi1 (plus Gi3). Gi2 and Go was studied in the olfactory epithelium and the main olfactory bulb of rats, using purified antibodies to the respective alpha subunits and beta gamma subunits of these G proteins. In the olfactory epithelium, only a restricted population of olfactory cells was immunopositive for Gi2 alpha, but others were not. The immunoreactivity for Gi1 alpha/Gi3 alpha was not observed. The olfactory epithelium was immunopositive for both Go alpha and beta gamma, but its apical surface was immunopositive only for beta gamma. In the main olfactory bulb, all layers were intensely immunopositive for Go alpha and beta gamma but weakly for Gi2 alpha. In contrast to the negative or weak immunostainings in the olfactory nerve fiber layer and glomeruli, the molecular and the internal granular layers were intensely immunopositive for Gi1 alpha/Gi3 alpha. These findings suggest the functional difference among Gi1/Gi3, Gi2 and Go in the signal transduction in the olfactory system.     

CD44 standard and variant isoform expression in normal human skin appendages and epidermis

 CD44 isoforms have been implicated in tumor progression and metastasis formation. This study presents a thorough immunohistochemical analysis of CD44 standard and isoform expression in normal human skin appendages and epidermis applying monoclonal antibodies against CD44s, CD44v3, -v4, -v5, -v6, and -v9. An improved immunohistochemical protocol with microwave-based antigen retrieval in paraffin sections and heavy metal amplification of the diaminobenzidine reaction product provided enhanced resolution and sensitivity as compared to studies on frozen sections. The hair follicle, the seborrheic and eccrine sweat glands were strongly positive for all CD44 isoforms studied. In the latter, the clear cells but not the dark (intercalated) cells were positive. The sudoriferous ducts adjacent to the glands were weakly positive for all CD44 isoforms and strongly positive near the skin surface. In the apocrine glands, the basal cells showed only a moderate positivity. The myoepithelial cells expressed only CD44s. In the epidermis, all CD44 isoforms were detectable, with strongest CD44 immunostaining in the lower third of the stratum spinosum and weaker staining in the stratum basale and the upper two-thirds of the stratum granulosum. The stratum granulosum and corneum were unreactive. Thus, a regional and cell type-specific CD44 expression was revealed. Accepted: 10 May 1996