Pavel Dráber

Plant γ-Tubulin Interacts with αβ-Tubulin Dimers and Forms Membrane-Associated Complexes

γ-Tubulin is assumed to participate in microtubule nucleation in acentrosomal plant cells, but the underlying molecular mechanisms are still unknown. Here, we show that γ-tubulin is present in protein complexes of various sizes and different subcellular locations in Arabidopsis and fava bean. Immunoprecipitation experiments revealed an association of γ-tubulin with αβ-tubulin dimers. γ-Tubulin cosedimented with microtubules polymerized in vitro and localized along their whole length. Large γ-tubulin complexes resistant to salt treatment were found to be associated with a high-speed microsomal fraction. Blue native electrophoresis of detergent-solubilized microsomes showed that the molecular mass of the complexes was >1 MD. Large γ-tubulin complexes were active in microtubule nucleation, but nucleation activity was not observed for the smaller complexes. Punctate γ-tubulin staining was associated with microtubule arrays, accumulated with short kinetochore microtubules interacting in polar regions with membranes, and localized in the vicinity of nuclei and in the area of cell plate formation. Our results indicate that the association of γ-tubulin complexes with dynamic membranes might ensure the flexibility of noncentrosomal microtubule nucleation. Moreover, the presence of other molecular forms of γ-tubulin suggests additional roles for this protein species in microtubule organization.

Production and characterization of a monoclonal antitubulin antibody.

Abstract A permanent hybridoma that secretes monoclonal antibody to tubulin has been developed. Monoclonal antibody belongs to the IgG1 subclass and its specificity was determined by enzymoimmunoassay and by indirect immunofluorescence. A cytoplasmic microtubule complex was detected by this monoclonal antibody as demonstrated in three different cell types.

Expression of class III beta-tubulin in normal and neoplastic human tissues.

Abstract The class III β-tubulin isotype is widely used as a neuronal marker in normal and neoplastic tissues. This isotype was, however, also immunodetected in certain tumours of non-neuronal origin such as squamous cell carcinoma. Using a newly described monoclonal antibody we compared the distribution of class III β-tubulin in normal and neoplastic tissues. The TU-20 mouse monoclonal antibody was prepared against a conserved synthetic peptide from the C-terminus of the human class III β-tubulin isotype, and its specificity was confirmed by immunoblotting, by competitive enzyme-linked immunosorbent assay and by immunofluorescence microscopy on cultured cells. In different cell lines of various origins the antibody reacted only with neuroblastoma Neuro-2a cells and with embryonal carcinoma P19 cells stimulated to neuronal differentiation by retinoic acid. Immunohistochemistry on formaldehyde-fixed paraffin-embedded normal human tissues revealed the presence of the class III β-tubulin isotype in cell bodies and processes of neuronal cells in the peripheral and central nervous systems. In other tissues, this β-tubulin isotype was not immunodetected. Class III β-tubulin was found in all cases of ganglioneuroblastoma, ganglioneuroma, medulloblastoma, neuroblastoma, sympathoblastoma and in one case of teratoma. In contrast, no reactivity was detected in tumours of non-neuronal origin, including 32 cases of squamous cell carcinoma. The results indicate a specific TU-20 epitope expression exclusively in neuronal tissues. The antibody could thus be a useful tool for the probing of class III β-tubulin functions in neurons as well as for immunohistochemical characterisation of tumours of neuronal origin.

Stability of monoclonal IgM antibodies freeze-dried in the presence of trehalose

We describe the use of the disaccharide trehalose for stabilization of mouse monoclonal IgM antibodies during freeze-drying and prolonged storage at elevated temperatures. Spent culture media, ascitic fluids and isolated immunoglobulins were freeze-dried in the presence of trehalose, stored at different temperatures, and tested after rehydration for their binding to their corresponding antigens. Antibodies, directed against various types of antigens, effectively recovered their binding efficiency as tested in enzyme-linked immunoassays, flow cytometry and immunofluorescence. Application of trehalose for freeze-drying of labile monoclonal IgM antibodies permits convenient long-term storage of large quantities of antibodies, facilitates their transport at ambient temperature and simplifies the construction of pre-aliquoted kits based on such antibodies.

Class III β-Tubulin Is Constitutively Coexpressed With Glial Fibrillary Acidic Protein and Nestin in Midgestational Human Fetal Astrocytes: Implications for Phenotypic Identity

Class III &bgr;-tubulin isotype (&bgr;III-tubulin) is widely regarded as a neuronal marker in developmental neurobiology and stem cell research. To test the specificity of this marker protein, we determined its expression and distribution in primary cultures of glial fibrillary acidic protein (GFAP)-expressing astrocytes isolated from the cerebral hemispheres of 2 human fetuses at 18 to 20 weeks of gestation. Cells were maintained as monolayer cultures for 1 to 21 days without differentiation induction. By immunofluorescence microscopy, coexpression of &bgr;III-tubulin and GFAP was detected in cells at all time points but in spatially distinct patterns. The numbers of GFAP+ cells gradually decreased from Days 1 to 21 in vitro, whereas &bgr;III-tubulin immunoreactivity was present in 100% of cells at all time points. &bgr;-III-tubulin mRNA and protein expression were demonstrated in cultured cells by reverse-transcriptase-polymerase chain reaction and immunoblotting, respectively. Glial fibrillary acidic protein+/&bgr;-III-tubulin-positive cells coexpressed nestin and vimentin but lacked neurofilament proteins, CD133, and glutamate-aspartate transporter. Weak cytoplasmic staining was detected with antibodies against microtubule-associated protein 2 isoforms. Confocal microscopy, performed on autopsy brain samples of human fetuses at 16 to 20 gestational weeks, revealed widespread colocalization of GFAP and &bgr;III-tubulin in cells of the ventricular/subventricular zones and the cortical plate. Our results indicate that in the midgestational human brain, &bgr;III-tubulin is not neuron specific because it is constitutively expressed in GFAP+/nestin+ presumptive fetal astrocytes.

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 dotlike 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 posttranslationally modified and that these modifications participate in the generation of plant tubulin polymorphism.

γ-tubulin redistribution in taxol-treated mitotic cells probed by monoclonal antibodies

Monoclonal antibodies were prepared against conserved synthetic peptide from the C-terminus of the gamma-tubulin and their specificity was confirmed by immunoblotting, competitive enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The antibodies decorated interphase centrosomes as well as half-spindles and midbodies in mitotic cells of various origin. The prepared antibodies were used to study the gamma-tubulin distribution in nocodazole and taxol-treated cells. In the cells recovering from the nocodazole treatment, gamma-tubulin was found in centers of all microtubule asters. Examination of relative location of gamma-tubulin and microtubule asters in taxol-treated mitotic cells 3T3, HeLa and PtK2 revealed that the number of taxol-induced microtubule asters exceeded the number of gamma-tubulin-positive spots. The gamma-tubulin was often found in the periphery of microtubule asters. Centrosomal phosphoprotein epitope detected by MPM-2 antibody colocalized with gamma-tubulin in taxol-treated mitotic cells. The presented data suggest that taxol-induced microtubule asters are in vivo nucleated independently of gamma-tubulin, and other minus-end nucleator(s) are necessary for formation of such asters. Alternatively, gamma-tubulin is present in subthreshold amounts undetectable by immunofluorescence.

Regulation of Microtubule Formation in Activated Mast Cells by Complexes of γ-Tubulin with Fyn and Syk Kinases

Aggregation of the high-affinity IgE receptors (FcεRIs) on the surface of granulated mast cells initiates a chain of signaling events culminating in the release of allergy mediators. Although microtubules are involved in mast cell degranulation, the molecular mechanism that controls microtubule rearrangement after FcεRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate leads to a rapid polymerization of microtubules. This polymerization was not dependent on the presence of Lyn kinase as determined by experiments with BMMCs isolated from Lyn-negative mice. One of the key regulators of microtubule polymerization is γ-tubulin. Immunoprecipitation experiments revealed that γ-tubulin from activated cells formed complexes with Fyn and Syk protein tyrosine kinases and several tyrosine phosphorylated proteins from both wild-type and Lyn−/− BMMCs. Pretreatment of the cells with Src-family or Syk-family selective tyrosine kinase inhibitors, PP2 or piceatannol, respectively, inhibited the formation of microtubules and reduced the amount of tyrosine phosphorylated proteins in γ-tubulin complexes, suggesting that Src and Syk family kinases are involved in the initial stages of microtubule formation. This notion was corroborated by pull-down experiments in which γ-tubulin complex bounds to the recombinant Src homology 2 and Src homology 3 domains of Fyn kinase. We propose that Fyn and Syk kinases are involved in the regulation of binding properties of γ-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.

Nuclear γ-Tubulin during Acentriolar Plant Mitosis

Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that γ-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of γ-tubulin in nuclei increased during the G2 phase, when cells are synchronized or sorted for particular phases of the cell cycle. γ-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear γ-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of γ-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.

Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis.

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhesive disk, median body, funis, mitotic spindles). MTOCs of the individual systems have not been fully defined. By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase and mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extracts the antibodies bound to a single polypeptide of approximately 50 kDa. Immunostaining of the interphase cell demonstrated gamma-tubulin as four bright spots at the basis of four out of eight flagella. Gamma-tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pairs of flagella which are formed de novo during cell division. Basal body regions of the anterolateral and caudal pairs of flagella which persist during the division and are integrated into the flagellar systems of the daughter cells did not show gamma-tubulin staining. At early mitosis, gamma-tubulin spots disappeared reappearing again at late mitosis in accord with reorientation of parent flagella and reorganization of flagellar apparatus during cell division. The antibody-detectable gamma-tubulin epitope was absent at the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin staining pattern thus confirming that the fluorescent label is exclusively located in the basal body regions. Our results point to a role of gamma-tubulin in nucleation of microtubules of newly formed flagella and indicate unusual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.