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).     

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.     

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     

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.     

Identification of multiple tubulins in taxol microtubules purified from carrot suspension cells

Tubulin has been purified from carrot suspension cells by ion-exchange chromatography and assembled into microtubules in the presence of 20 microM taxol. One-dimensional SDS-PAGE suggested that the alpha band migrated faster than the beta band (as has been established for some lower eukaryotic tubulins) and this heterology with brain tubulins was confirmed by peptide mapping. When subjected to two-dimensional gel electrophoresis, the plant tubulins could be separated into multiple alpha and beta isotypes. Immunoblotting, using monoclonal anti-tubulins, confirmed that the tubulin isotypes identified in taxol microtubules represent all of the tubulins present in homogenates of unsynchronised log-phase carrot suspension cells. All identified tubulins are therefore assembly-competent under these conditions. Plant cells can contain four different microtubule arrays, but cells arrested in G0/G1 contain only cortical microtubule arrays; such cells, however, exhibit the same tubulin profile as non-synchronised cells, thereby showing no restriction in the number of subunits during this phase of the cell cycle.     

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     

Differential subcellular distribution of tubulin epitopes in boar spermatozoa: recognition of class III beta-tubulin epitope in sperm tail

The exposure of tubulin epitopes was studied in ejaculated boar spermatozoa using a panel of four monoclonal antibodies specific to the N-terminal or C-terminal structural domains of tubulin and three monoclonal antibodies against class III beta-tubulin. The specificity of the antibodies was confirmed by immunoblotting. Immunocytochemical staining showed that antibodies discriminated between various parts of a spermatozoon, and that epitopes of class III beta-tubulin were present in the flagellum. A tubulin epitope from the C-terminal domain of beta-tubulin was detected in the triangular segment of the postacrosomal part of the sperm head. Its distribution changed after an A23187 ionophore-induced acrosome reaction, indicating that tubulin participates in the early stages of fertilization. Three monoclonal antibodies, TU-20, SDL.3D10, and TUJ1 directed against epitopes on the C-terminal end of neuron-specific class III beta-tubulin that is widely used as a neuronal marker, stained the flagella. The reactivity of TU-20 was further confirmed by absorbing the antibody with the immunizing peptide and by immunoelectron microscopy. Immunoblotting after two-dimensional electrophoresis revealed that the corresponding epitope was not present on all beta-tubulin isoforms. These results suggest that various tubulins are involved in the functional organization of the mammalian sperm flagellum and head.     

Serological similarity of flagellar and mitotic microtubules

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.     

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     

Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium

Summary— Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the α N-terminal domain, one in the β C-terminal one, and two in α and β central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of α-tubulin whereas the epitopes of three other ones (TAP 952°, AXO 58 and AXO 49°) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both α- and β-tubulins. The AXO 49° specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.     

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 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.     

Inhibition of microtubule assembly in vitro by anti-tubulin monoclonal antibodies

Five monoclonal antibodies against N-terminal domains of alpha- or beta-tubulin were tested for their ability to interfere with the in vitro formation of microtubules. Although all the antibodies exhibited similar association constants for immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. For the most potent antibody, TU-13, the antibody/tubulin molar ratio of about 1:320 was sufficient for a 50% inhibition. The data indicate that the surface regions of N-terminal domains of tubulin are involved in the formation of microtubules.     

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.     

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

Summary 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.     

Distinct localization of a beta-tubulin epitope in the Tetrahymena thermophila and Paramecium caudatum cortex

Summary. Many of the highly organized microtubular arrangements in ciliates are located in the cortical area containing membrane vesicles and vacuoles. In Tetrahymena thermophila and Paramecium caudatum, immunofluorescence microscopy with the monoclonal antibody TU-06, directed against β-tubulin, revealed distinct staining of this cortical region alone, while the cilia and other microtubular structures were unstained. The specificity of the antibody was confirmed by immunoblotting and by preabsorption of the antibody with purified tubulin. Double-label immunofluorescence with antibodies against γ-tubulin, detyrosinated α-tubulin, and centrin showed that the TU-06 epitope is localized outside the basal body region. This was also confirmed by immunogold electron microscopy of thin sections. Proteolytic digestion of porcine brain β-tubulin combined with a peptide scan of immobilized, overlapping peptides disclosed that the epitope was in the β-tubulin region β81–95, a region which is phylogenetically highly conserved. As known posttranslational modifications of β-tubulin are located outside this area, the observed staining pattern cannot be interpreted as evidence of subcellular sequestration of modified tubulin. The limited distribution of the epitope could rather reflect the dependence of TU-06 epitope exposition on conformations of tubulin molecules in microtubule arrangements or on differential masking by interacting proteins. Correspondence and reprints: Institute of Molecular Genetics, Czech Academy of Sciences, Vídeňská 1083, 14220 Prague, Czech Republic.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

Spatial distribution of posttranslationally modified tubulins in polarized cells of developing Artemia

In many differentiated cells, posttranslationally modified tubulins exhibit restricted subcellular distribution, leading to the proposal that they are required for the production and maintenance of polarity. To study this possibility, we used immunological approaches to examine tubulin isoforms in developing Artemia larvae and to determine their location in several types of cells within the organism. The amount of tubulin in relation to total protein remained relatively constant during early larval development while detyrosinated tubulin increased, an event correlated with the differentiation of larval gut muscle cells. Except for epidermal cells of the developing thorax, each type of cell within the Artemia larvae exhibited characteristic staining patterns which were very similar for each antitubulin antibody. Within epidermal cells, microtubules containing acetylated tubulin appeared patchy or punctate in their distribution, an image not seen with the other antibodies. In most polarized cells, staining for tubulin and actin colocalized in discrete areas, demonstrating enrichment of both proteins within the same cellular compartment and suggesting functional interactions. Mitotic figures were stained with qualitatively equal intensity by all of the antitubulin antibodies, but asters were not observed. Midbodies were intensely stained with phalloidin as well as the antibodies to tubulin. It was clear that microtubules exhibited a preferential localization in cells of Artemia but in no case was a tubulin isoform found exclusively in one area of a cell. The results support the contention that microtubules influence the organization of polarized cell structure and function but they do not permit the conclusion that this capability is dependent on the localization of posttranslationally modified tubulins to restricted subcellular positions.     

Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.     

Microtubules in bacteria: Ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as "bacterial microtubules" (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening.