Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of alpha-tubulin.

The state of tubulin tyrosination in the fission yeast Schizosaccharomyces pombe was investigated using a combination of indirect immunofluorescence microscopy and Western blotting. Antibodies specific for the tyrosinated form of alpha-tubulin stained all microtubule arrays in wild type cells and recognised the two alpha-tubulin polypeptides in Western blots of cell extracts enriched for tubulin by DEAE-Sephadex chromatography. Antisera that specifically recognised the detyrosinated, glu, form, on the other hand, gave consistently negative results, both in cells undergoing rapid exponential growth and in those allowed to accumulate in stationary phase. Neither the "ageing" of microtubules, by arresting cells at different points (late G1 or G2/M) in the cell division cycle, nor stabilising them, using D2O, lead to any detectable tubulin detryrosination. These results suggest that S. pombe lacks the carboxypeptidase that carries out the tubulin detyrosination reaction. This is the first report of an organism that possesses the correct C-terminal alpha-tubulin sequence yet fails to carry out this post-translational modification. The implication of this novel finding for the biological role of these events is discussed.     

A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. II. Effects on cell movement, organization of microtubules, and intermediate filaments, and arrangement of Golgi elements

A rat monoclonal antibody against yeast alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) that reacts specifically with the tyrosylated form of alpha- tubulin and readily binds to tubulin in microtubules when injected into cultured cells (see Wehland, J., M. C. Willingham, and I. V. Sandoval, 1983, J. Cell Biol., 97:1467-1475) was used to study microtubule organization and function in living cells. Depending on the concentration of YL 1/2 that was injected the following striking effects were observed: (a) When injected at a low concentration (2 mg IgG/ml in the injection solution), where microtubules were decorated without changing their distribution, intracellular movement of cell organelles (saltatory movement) and cell translocation were not affected. Intermediate concentrations (6 mg IgG/ml) that induced bundling but no perinuclear aggregation of microtubules abolished saltatory movement and cell translocation, and high concentrations (greater than 12 mg IgG/ml) that induced perinuclear aggregation of microtubules showed the same effect. (b) YL 1/2, when injected at intermediate and high concentrations, arrested cells in mitosis. Such cells showed no normal spindle structures. (c) Injection of an intermediate concentration of YL 1/2 that stopped saltatory movement caused little or no aggregation of intermediate filaments and no dispersion of the Golgi complex. After injection of high concentrations, resulting in perinuclear aggregation of microtubules, intermediate filaments formed perinuclear bundles and the Golgi complex became dispersed analogous to results obtained after treatment of cells with colcemid. (d) When rhodamine-conjugated YL 1/2 was injected at concentrations that stopped saltatory movement and arrested cells in mitosis, microtubule structures could be visualized and followed for several hours in living cells by video image intensification microscopy. They showed little or no change in distribution and organization during observation, even though these microtubule structures appeared not to be stabilized by injected YL 1/2 since they were readily depolymerized by colcemid or cold treatment and repolymerized upon drug removal or rewarming to 37 degrees C, respectively. These results are discussed in terms of the participation of microtubules in cellular activities such as cell movement and cytoplasmic organization and in terms of the specificity of YL 1/2 for the tyrosylated form of alpha-tubulin.     

Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells

Cells were prepared for indirect immunofluorescence microscopy after paraformaldehyde fixation of multicellular root apices and brief incubation in cell wall-digesting enzymes. This allowed subsequent separation of the tissue into individual cells or short files of cells which were put onto coverslips coated with polylysine. Unlike spherical protoplasts made from living tissues, these preparations retain the same polyhedral shape as the cells from which they are derived. Cellular contents, including organized arrays of microtubules, are likewise structurally stabilized. Antibodies to porcine brain tubulin react with all types of microtubule array known to occur in plant meristematic cells, namely, interphase cortical microtubules, pre- prophase bands, the mitotic spindle, and phragmoplast microtubules. The retention of antigenicity in permeabilized, isolated, stabilized cells from typical, wall-enclosed plant cells has much potential for plant immunocytochemistry, and in particular should facilitate work on the role of microtubules in the morphogenesis of organized plant tissues.     

A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and microtubule polymerization and organization in vivo

The antigenic site recognized by a rat monoclonal antibody (clone YL 1/2) reacting with alpha-tubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) has been determined and partially characterized. YL 1/2 reacts specifically with the tyrosylated form of brain alpha-tubulin from different mammalian species. YL 1/2 reacts with the synthetic peptide Gly-(Glu)3-Gly-(Glu)2- Tyr, corresponding to the carboxyterminal amino acid sequence of tyrosylated alpha-tubulin, but does not react with Gly-(Glu)3-Gly- (Glu)2, the constituent peptide of detyrosylated alpha-tubulin. Electron microscopy as well as direct and indirect immunofluorescence microscopy shows that YL 1/2 binds to the surface of microtubules polymerized in vitro and in vivo. Further in vitro studies show that the antibody has no effect on the rate and extent of microtubule polymerization, the stability of microtubules, and the incorporation of the microtubule-associated proteins (MAP2) and tau into microtubules. In vivo studies using Swiss 3T3 fibroblasts injected with YL 1/2 show that; when injected at low concentration (2 mg IgG/ml in the injection solution), the antibody binds to microtubules without changing their distribution in the cytoplasm. Injection of larger concentration of YL 1/2 (6 mg IgG/ml) induces the formation of microtubule bundles, and still higher concentrations cause the aggregation of microtubule bundles around the nucleus (greater than 12 mg IgG/ml).     

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.     

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.     

Characterization of a monoclonal antibody reacting with histone H3

A hybridoma cell line, 1GB3, has been obtained from a fusion between SP/O-Ag 14 myeloma cells and lymphocytes from BALB/c mice immunized with rat liver nuclear proteins. This hybridoma secreted a monoclonal antibody of the IgG2b class which reacted specifically with histone H3 in enzyme-linked immunosorbent assay (ELISA) as well as in immunoblotting and immunodot assays. Stringent test conditions were necessary to eliminate the presence of nonspecific or contaminating reactions with other histones than H3. The monoclonal antibody appears to recognize an epitope situated in the N-terminal residues 20-50 of histone H3; it recognizes this epitope in the octamer aggregate of core histones but not in the core particle.     

Perisynaptic Schwann cells at neuromuscular junctions revealed by a novel monoclonal antibody

This study aimed to generate a probe for perisynaptic Schwann cells (PSCs) to investigate the emerging role of these synapse-associated glial cells in the formation and maintenance of the neuromuscular junction (NMJ). We have obtained a novel monoclonal antibody, 2A12, which labels the external surface of PSC membranes at the frog NMJ. The antibody reveals PSC fine processes or “fingers” that are interposed between nerve terminal and muscle membrane, interdigitating with bands of acetylcholine receptors. This antibody also labels PSCs at the avian neuromuscular junction and recognizes a 200 kDa protein in Torpedo electric organs. In frog muscles, axotomy induces sprouting of PSC processes beyond clusters of acetylcholine receptors and acetylcholinesterase at denervated junctional branches. PSC branches often extend across several muscle fibers. At some junctions, PSC sprouts join the tips of neighboring branches. The average length of PSC sprouts is approximately 156 µ at 3-week denervated NMJs. PSC sprouting is accompanied by a significant increase in the number of Schwann cell bodies per NMJ. Following nerve regeneration, nerve terminals reinnervate the junction along the PSC processes. In vivo observations of normal frog muscles also show PSC processes longer than nerve terminals at some junctional branches. The results suggest that nerve injury induces profuse PSC sprouting that may play a role in guiding nerve terminal regeneration at frog NMJs. In addition, antibody 2A12 reveals the fine morphology of PSCs in relation to other synaptic elements and is a useful probe in elucidating the function of these synapse-associated glial cells in vivo.     

An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha-tubulin

Rhodamine-labeled monoclonal antibodies, which react with tyrosinated alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) and label microtubules in vivo (Wehland, J., M. C. Willingham, and I. Sandoval, 1983, J. Cell Biol., 97:1467-1475) were microinjected into syncytial stage Drosophila embryos. At 1 mg/ml antibody concentration, the microtubule arrays of the surface caps became labeled by YL 1/2 but normal development was found to continue. The results are compared with the data from fixed material particularly with regard to interphase microtubules, centrosome separation, and spindle and midbody formation. At 5 mg/ml antibody concentration the microtubules took up larger quantities of antibodies and clumped around the nuclei. Nuclei with clumped microtubules lost their position in the surface layer and moved into the interior. As a result, the F-actin cap meshwork associated with such nuclei either failed to form or subsided. It is concluded that microtubule activity is required to maintain the nuclei in the surface layer and organize the F-actin meshwork of the caps.     

Characterization of monoclonal antibody fragments produced by plant cells

Production of a murine IgG1 was investigated using hairy roots, shooty teratomas, and suspended cells of transgenic tobacco. In all cases, in addition to complete assembled antibody, two to four major antibody fragments accumulated in the biomass. A range of protease inhibitors, protein-stabilizing agents, inhibitors of N-glycosylation and protein secretion, glycan-reactive agents, and affinity probes was used to characterize these fragments and investigate their sites and mechanisms of formation. The fragments were not experimental artifacts caused by antibody degradation during tissue homogenization and sample preparation, nor did they represent glycosylation variants. All of the molecules were actively secreted into the culture media and some showed evidence of Golgi-associated glycan processing, indicating they were not assembly intermediates. Antibody fragments of 50 and 80 kDa were identified mainly as the products of extracellular degradation in the root and shoot apoplast; the 80-kDa fragment was also present in cell suspension medium, and in suspended cell biomass toward the end of the growth phase. Larger 120- and 135-kDa fragments were most likely produced by proteolytic degradation along the secretory pathway outside of the endoplasmic reticulum (ER) and Golgi apparatus; the carbohydrate residues of the 135-kDa antibody suggest formation between these organelles. Inhibition of protein secretion and retention of antibody in the ER and/or Golgi reduced fragmentation and increased antibody accumulation levels, probably by reducing exposure to the principal sites of protease activity. This work highlights the importance of foreign protein degradation in plant tissues as a mechanism for posttranslational product loss. Identifying the nature of these degradative processes is a first step toward alleviating their effects, improving protein yields, and enhancing the feasibility of plants as a commercial means for large-scale protein production.     

Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody

A monoclonal antibody (CC-212), obtained in a fusion experiment in which basal bodies from quail oviduct were used as immunogen, has been shown to label the apical pole of ciliated cells and to react with a 200-kD protein. This monoclonal antibody was demonstrated to be an anti-myosin from smooth muscle or from nonmuscular cells using the following criteria: On Western blots it reacted with the myosin heavy chains from gizzard and platelet extracts and from cultured cell line extracts, but did not react with striated muscle myosin heavy chains. By immunofluorescence it decorated the stress fibers of well-spread cells with a characteristic striated pattern, while it did not react with myotubes containing organized myofibrils. On native ciliated cells as well as on Triton-extracted ciliated cortices from quail oviduct, this monoclonal antibody decorated the apical pole with a stronger labeling of the periphery of the apical area. Ultrastructural localization was attempted using the immunogold technique on the same preparation. Myosin was associated with a filamentous material present between striated rootlets and the proximal extremities of the basal bodies. No labeling of the basal body itself or of axoneme was observed.     

FB1, a monoclonal antibody reacting with a keratin 14 epitope, stains only a small subset of psoriatic basal keratinocytes

Abstract
A murine monoclonal antibody, FB1, reacted with the basal keratinocytes of human stratified epithelia. One-dimensional and two-dimensional immunoblotting assays, performed on keratins extracted from HaCat cells and normal human keratinocytes, showed that FBI recognizes K14. When LL002, another K14 monoclonal antibody is added, the FB1 stained area in the 2D-immunoblot seems to cover a fraction of the LL002 spot. Immunohistochemical data obtained from studies on normal human tissues supported the K14 specificity of FB1, but when compared with two other monoclonal antibodies, LL002 and RCK107 reacting with K14, some differences appeared. These differences were mainly seen in sweat glands, hair follicles, psoriatic epidermis and salivary glands. In psoriatic epidermis, FB1 showed a heterogeneous pattern of staining of the basal cell compartment. Intense reactivity was only observed at the bottom of the rete ridges. Staining diminished and finally disappeared in the basal cells above the dermal papillae. This observation supports the view that an increased germinative cell population in psoriasis involves a partially differentiated amplifying compartment in which the number of cell divisions is increased.     

A subpopulation of trypanosome microtubules recognized by a monoclonal antibody to tubulin

Two hybridoma cell lines were selected after the fusion of the myeloma cell line X-63 Ag8-653 with spleen cells from mice immunized with bovine brain microtubules. These lines, clones 3F3 and 16D3, secrete IgM antibodies both staining a fibrillar network in fibroblasts. Autoradiography of immunoblots of SDS gels showed that the antigenic determinants defined by these antibodies are present on tubulin and also on several other polypeptides in mammalian cells. In contrast, they were found to react only with tubulin in Trypanosoma brucei, parasitic protozoan which are the causative agent of sleeping sickness. By immunofluorescence microscopy, 3F3 bound only to a subpopulation of microtubules associated with the flagellum of these cells when, under the same conditions, 16D3 stained other microtubule populations including sub-pellicular microtubules. These results show that flagellar tubulin differs from tubulin of other locations in the same cell by at least one antigenic determinant which could be involved in microtubule specialization.     

Inhibition of iodothyronine transport into rat liver cells by a monoclonal antibody

The role of the rat liver plasma membrane in the regulation of uptake and subsequent deiodination of thyroxine (T4) or the biologically active thyroid hormone 3,3',5-triiodothyronine (T3) was investigated. Here we report on the production of monoclonal antibodies raised against rat hepatocytes. Two antibodies were selected. Antibody ER-22 did bind to a Mr 52,000 membrane protein and inhibited the 1- and 5-min uptake of both T4 and T3 by primary cultured rat hepatocytes in a dose-dependent fashion. As the uptake of T4 and T3 depends on the presence of a sodium gradient over the plasma membrane, the inhibitory potency of ER-22 on the Na+,K+-ATPase activity was investigated. No inhibition of the uptake of 86Rb+ could be determined, indicating that antibody ER-22 is not directed against the Na+,K+-ATPase but probably the carrier protein itself. Clearance of T3 from the medium and concomitant iodide production by cultured rat hepatocytes during a 20-h incubation in the presence of ER-22 were both inhibited by 50% with respect to a control incubation in the absence of monoclonal antibody, pointing to the importance of carrier-mediated transport in cellular uptake and metabolism of T3. A second monoclonal antibody did bind to two other plasma membrane proteins but did not inhibit transport of thyroid hormone.     

A conformational transition between an open and closed form of human pancreatic lipase revealed by a monoclonal antibody

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. Here we observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds. A single conformational mAb (248-31) fails to immunoprecipitate HPL in the presence of activator compounds and HPL covalently inhibited with diethyl p-nitrophenyl phosphate (DP.HPL). This loss of recognition of HPL by mAb 248-31 was probably due to the motion of the lid, since HPL(-lid) was always recognized in the presence or absence of activator compounds. Furthermore, two other mAbs (81-23 and 146-40) immunoprecipitated HPL similarly whether or not the activator compounds were present. MAb 248-31 therefore specifically recognizes HPL in the closed but not the open conformation.     

Novel polysaccharide antigen of Orientia tsutsugamushi revealed by a monoclonal antibody

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates in the cytosol of host cells. Although several protein antigens have been characterized and cloned, little information exists regarding the polysaccharide antigen of this bacterium. In this study, we identified and characterized a novel antigen defined by a monoclonal antibody (MAb), NT19, against O. tsutsugamushi . Immunofluorescence microscopic studies showed that the NT19 antigen is released from the bacteria in the cytosol of host cells forming aggregates with bacteria. Immunoblot analysis showed that MAb NT19 recognized a strong band with a molecular mass of 20 kDa that was resistant to proteinase K digestion and sensitive to periodate oxidation, suggesting that the NT19 antigen is a polysaccharide. The function of this polysaccharide is not known, but considering its distribution within a bacterial microcolony, it is suspected to be involved in forming a biofilm-like structure within host cells.     

Differential expression of alpha-tubulin mRNA in rat cerebellum as revealed by in situ hybridization

Nucleotide sequence analysis of two rat alpha-tubulin cDNA clones showed a marked divergence in their 3'-untranslated regions. However, each of the alpha-tubulin isotypes shows a high interspecies homology in this region, when compared with an isotubulin sequence from human and Chinese hamster. In situ hybridization of rat cerebellum with alpha-tubulin cDNA revealed differential expression in various cell layers. The mitotically active cells in the external granular layer show the highest level of alpha-tubulin mRNA, while lower levels are observed in the migrating cells in the molecular layer and in the differentiating cells in the internal granular layer. Very low levels of the mRNA are observed in the prenatally differentiated Purkinje cells.     

Production of a monoclonal antibody reacting with vimentin, a structural protein of intermediate filaments

This paper reports on the production of a monoclonal antibody (i-18) reacting with vimentin, the major structural component of intermediate filaments in cells of mesenchymal origin. The antibody was obtained following immunization with hamster fibroblasts and was selected for its ability to bind to the cytoskeleton fraction of the aforementioned cells. It decorated a perinuclear filamentous network characteristic of vimentin filaments in cells of mesenchymal origin of avian through human species. The specificity of the reagent was further ascertained on the basis of the sensitivity of the decorated filaments to colcemide. The strict antibody specificity for cells of mesenchymal versus epithelial origin was confirmed also in vivo on histological specimens from solid tissue. The i-18 monoclonal antibody precipitated a molecule of about 57 Kd from metabolically labelled cellular extracts. The broad cross-reactivity of this monoclonal antibody among different animal species, as well as its strict in vivo mesenchymal tissue specificity makes this antibody a useful reagent for both experimental and diagnostic purposes.     

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.     

The aminoterminal domain of alpha-tubulin probed by monoclonal antibodies. Recognition of a rarely exposed epitope by the monoclonal antibody 111 B52 C2

The production and identification of a monoclonal antibody, 111 B52 C2, raised against fragments obtained after limited proteolysis of purified tubulin is described. The recognized epitope is located on the aminoterminal domain of the alpha-tubulin subunit and differs from the antigenic sites reacting with the presently existing panel of available monoclonal antibodies. This monoclonal antibody thus constitutes a potentially useful tool to explore interactions between tubulin and other specific ligands.