BACKGROUND: Poor semen quality is one of the main causes of infertility. We have generated a set of monoclonal antibodies to human sperm and used them to investigate sperm quality. Some of these antibodies found differences in the expression of proteins between normal sperm and pathological sperm displaying severe defects. One of them was the Hs-14 antibody. The aim of this paper was to determine the target protein of the Hs-14 monoclonal antibody and to investigate the expression of the Hs-14-reacting protein on the sperm of asthenozoospermic men with sperm motility defect and of healthy normozoospermic men. METHODS: Indirect immunofluorescence, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry. RESULTS: The Hs-14 antibody binds fibronectin, β-tubulin and valosin-containing protein - new name for this protein is transitional endoplasmic reticulum ATPase (TERA). Since the Hs-14 reaction with TERA remained the strongest at the highest antibody dilution, and Hs-14 consistently labelled the same spot or band as the monospecific anti-TERA antibody on immunoblots, we assume that TERA is an Hs-14-specific protein. Binding of fibronectin and β-tubulin might represent nonspecific cross-reactivity or Hs-14 reaction with similar epitopes of these proteins. A significant difference (P < 0.001) in immunofluorescence staining with Hs-14 was found between the normozoospermic and asthenozoospermic men. CONCLUSION: The Hs-14 antibody enables discrimination between sterile or subfertile asthenozoospermic and fertile normozoospermic men. Decreased levels of TERA in men can be used as a biomarker of reduced fertility.

INTRODUCTION: La pauvre qualité de la semence est l’une des causes d’infertilité. Nous avons généré une série d’anticorps monoclonaux contre le sperme humain et nous l’avons utilisée pour examiner la qualité du sperme. Certains de ces anticorps ont montré des différences d’ expression des protéines entre le sperme normal et le sperme pathologique qui a des défauts sévères. L’un d’eux a été l’anticorps Hs-14. Le but de cet article était de déterminer la protéine cible de l’anticorps monoclonal Hs-14 et d’établir l’expression de la protéine réagissant avec Hs-14 sur le sperme des hommes asthénozoospermiques qui ont des défauts de la mobilité du sperme et sur celui des hommes normozoospermiques. MÉTHODES: Immunofluorescence indirecte, electrophorèse sur gel polyacrylamide à une ou deux dimensions, immunoblotting et spectrométrie de masse. RÉSULTATS: L’anticorps Hs-14 s’attache à la fibronectine, à la β-tubuline et à la protéine TERA (ATPase transitoire de réticulum endoplasmique). Etant donné que la réaction du Hs-14 avec TERA a été la plus forte à la dilution la plus grande de l’anticorps, et que Hs-14 marquait systématiquement la même tache ou bande que l’anticorps mono-spécifique anti-TERA sur les immunoblots, nous supposons que TERA est une protéine spécifique pour Hs-14. L’attachement à la fibronectine et à la β-tubuline pourrait représenter une réaction croisée non spécifique ou la réaction du Hs-14 avec des épitopes similaires de ces protéines. Une différence significative (P < 0.001) en immunofluorescence avec Hs-14 a été révélée entre hommes normozoospermiques et asthénozoospermiques. CONCLUSIONS: L’anticorps Hs-14 permet de différencier les hommes stériles ou subfertiles asthénozoospermiques des hommes fertiles normozoospermiques. Les niveaux de la TERA chez les hommes pourraient être utilisés comme un marqueur biologique d’une fertilité réduite.

• Keywords
• Acrosome, Asthenozoospermia, Human spermatozoa, Monoclonal antibody, Transitional endoplasmic reticulum ATPase,
• Publication type
• Journal Article MeSH

The U4/U6·U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) is an essential pre-mRNA splicing factor, which is assembled in a stepwise manner before each round of splicing. It was previously shown that the tri-snRNP is formed in Cajal bodies (CBs), but little is known about the dynamics of this process. Here we created a mathematical model of tri-snRNP assembly in CBs and used it to fit kinetics of individual snRNPs monitored by fluorescence recovery after photobleaching. A global fitting of all kinetic data determined key reaction constants of tri-snRNP assembly. Our model predicts that the rates of di-snRNP and tri-snRNP assemblies are similar and that ∼230 tri-snRNPs are assembled in one CB per minute. Our analysis further indicates that tri-snRNP assembly is approximately 10-fold faster in CBs than in the surrounding nucleoplasm, which is fully consistent with the importance of CBs for snRNP formation in rapidly developing biological systems. Finally, the model predicted binding between SART3 and a CB component. We tested this prediction by Förster resonance energy transfer and revealed an interaction between SART3 and coilin in CBs.

• MeSH
• Antigens, Neoplasm genetics   metabolism   MeSH
• Cell Nucleus genetics   metabolism   MeSH
• Coiled Bodies genetics   metabolism   MeSH
• HeLa Cells MeSH
• Nuclear Proteins metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear genetics   metabolism   MeSH
• Ribonucleoprotein, U5 Small Nuclear genetics   metabolism   MeSH
• Models, Molecular * MeSH
• Cell Line, Tumor MeSH
• RNA Precursors genetics   metabolism   MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Ribonucleoproteins, Small Nuclear genetics   metabolism   MeSH
• RNA Helicases genetics   metabolism   MeSH
• RNA Splicing genetics   MeSH
• Spliceosomes genetics   metabolism   MeSH
• Protein Binding genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Neoplasm MeSH
• DHX15 protein, human MeSH Browser
• Nuclear Proteins MeSH
• Ribonucleoprotein, U4-U6 Small Nuclear MeSH
• Ribonucleoprotein, U5 Small Nuclear MeSH
• p80-coilin MeSH Browser
• RNA Precursors MeSH
• RNA-Binding Proteins MeSH
• Ribonucleoproteins, Small Nuclear MeSH
• RNA Helicases MeSH
• SART3 protein, human MeSH Browser
• SNRNP200 protein, human MeSH Browser

BACKGROUND: The function of the cortical microtubules, composed of alphabeta-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited. RESULTS: Using homology modeling we have generated an Arabidopsis thaliana microtubule protofilament model that served for the prediction of surface exposure of five beta-tubulin epitopes as well as tyrosine residues. Peptide scans newly disclosed the position of epitopes detected by antibodies 18D6 (beta1-10), TUB2.1 (beta426-435) and TU-14 (beta436-445). Experimental verification of the results by immunofluorescence microscopy revealed that the exposure of epitopes depended on the mode of fixation. Moreover, homology modeling showed that only tyrosines in the C-terminal region of beta-tubulins (behind beta425) were exposed on the microtubule external side. Immunofluorescence microscopy revealed tyrosine phosphorylation of microtubules in plant cells, implying that beta-tubulins could be one of the targets for tyrosine kinases. CONCLUSIONS: We predicted surface exposure of five beta-tubulin epitopes, as well as tyrosine residues, on the surface of A. thaliana microtubule protofilament model, and validated the obtained results by immunofluorescence microscopy on cortical microtubules in cells.The results suggest that prediction of epitope exposure on microtubules by means of homology modeling combined with site-directed antibodies can contribute to a better understanding of the interactions of plant microtubules with associated proteins.

• MeSH
• Arabidopsis immunology   MeSH
• Epitopes immunology   MeSH
• Microscopy, Fluorescence MeSH
• Epitope Mapping methods   MeSH
• Microtubules immunology   MeSH
• Models, Molecular MeSH
• Antibodies, Monoclonal immunology   MeSH
• Arabidopsis Proteins immunology   MeSH
• Tubulin immunology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epitopes MeSH
• Antibodies, Monoclonal MeSH
• Arabidopsis Proteins MeSH
• Tubulin MeSH

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 beta-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 gamma-tubulin, detyrosinated alpha-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 beta-tubulin combined with a peptide scan of immobilized, overlapping peptides disclosed that the epitope was in the beta-tubulin region beta81-95, a region which is phylogenetically highly conserved. As known posttranslational modifications of beta-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.

• MeSH
• Cell Membrane immunology   MeSH
• 3T3 Cells MeSH
• Epitopes analysis   immunology   metabolism   MeSH
• Immunoblotting MeSH
• Epitope Mapping MeSH
• Mice MeSH
• Paramecium immunology   MeSH
• Tetrahymena thermophila immunology   MeSH
• Tubulin immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epitopes MeSH
• Tubulin MeSH

gamma-Tubulin is assumed to participate in microtubule nucleation in acentrosomal plant cells, but the underlying molecular mechanisms are still unknown. Here, we show that gamma-tubulin is present in protein complexes of various sizes and different subcellular locations in Arabidopsis and fava bean. Immunoprecipitation experiments revealed an association of gamma-tubulin with alphabeta-tubulin dimers. gamma-Tubulin cosedimented with microtubules polymerized in vitro and localized along their whole length. Large gamma-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 gamma-tubulin complexes were active in microtubule nucleation, but nucleation activity was not observed for the smaller complexes. Punctate gamma-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 gamma-tubulin complexes with dynamic membranes might ensure the flexibility of noncentrosomal microtubule nucleation. Moreover, the presence of other molecular forms of gamma-tubulin suggests additional roles for this protein species in microtubule organization.

• MeSH
• Antibodies, Antinuclear genetics   metabolism   MeSH
• Arabidopsis metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Cytosol metabolism   MeSH
• Dimerization MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Fluorescent Antibody Technique MeSH
• Microtubules metabolism   MeSH
• Microsomes metabolism   MeSH
• Mitosis physiology   MeSH
• Precipitin Tests MeSH
• Arabidopsis Proteins metabolism   MeSH
• Tubulin chemistry   immunology   metabolism   MeSH
• Protein Binding MeSH
• Vicia faba metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antibodies, Antinuclear MeSH
• Arabidopsis Proteins MeSH
• Tubulin MeSH

gamma-Tubulin is necessary for nucleation and polar orientation of microtubules in vivo. The molecular mechanism of microtubule nucleation by gamma-tubulin and the regulation of this process are not fully understood. Here we show that there are two gamma-tubulin forms in the brain that are present in complexes of various sizes. Large complexes tend to dissociate in the presence of a high salt concentration. Both gamma-tubulins co-polymerized with tubulin dimers, and multiple gamma-tubulin bands were identified in microtubule protein preparations under conditions of non-denaturing electrophoresis. Immunoprecipitation experiments with monoclonal antibodies against gamma-tubulin and alpha-tubulin revealed interactions of both gamma-tubulin forms with tubulin dimers, irrespective of the size of complexes. We suggest that, besides small and large gamma-tubulin complexes, other molecular gamma-tubulin form(s) exist in brain extracts. Two-dimensional electrophoresis revealed multiple charge variants of gamma-tubulin in both brain extracts and microtubule protein preparations. Post-translational modification(s) of gamma-tubulins might therefore have an important role in the regulation of microtubule nucleation in neuronal cells.

• MeSH
• Dimerization MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Cell Fractionation MeSH
• Brain Chemistry * MeSH
• Swine MeSH
• Protein Isoforms MeSH
• Tissue Extracts chemistry   metabolism   MeSH
• Tubulin chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protein Isoforms MeSH
• Tissue Extracts MeSH
• Tubulin MeSH

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 gamma-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of gamma-tubulin in nuclei increased during the G(2) phase, when cells are synchronized or sorted for particular phases of the cell cycle. gamma-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 gamma-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of gamma-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.

• MeSH
• Cell Nucleus chemistry   MeSH
• Cell Cycle MeSH
• Centrioles MeSH
• Fabaceae chemistry   metabolism   MeSH
• Fluorescent Antibody Technique MeSH
• Microscopy, Confocal MeSH
• Plants, Medicinal MeSH
• Mitosis * MeSH
• Plants chemistry   metabolism   MeSH
• Tubulin metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tubulin MeSH

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 delta 2-tubulin variants were detected on alpha-tubulin subunits; polyglutamylation was also found on beta-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 delta 2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and delta 2-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 alpha- and beta-tubulin molecules, respectively, revealed that 11 isoforms belonged to the alpha-subunit and 11 isoforms to the beta-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several alpha-tubulin isoforms, antibodies against nontyrosinated and delta 2-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.

• MeSH
• Acetylation MeSH
• Chromatography, Ion Exchange MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Genetic Variation MeSH
• Immunoblotting MeSH
• Isoelectric Focusing MeSH
• Plants, Toxic * MeSH
• Cells, Cultured MeSH
• Microtubules metabolism   ultrastructure   MeSH
• Antibodies, Monoclonal MeSH
• Mice MeSH
• Protein Processing, Post-Translational * MeSH
• Nicotiana metabolism   MeSH
• Tubulin analysis   isolation & purification   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antibodies, Monoclonal MeSH
• Tubulin MeSH

The expression of vimentin and the phosphorylated variant of high molecular weight neurofilament protein (NF-H) was studied in developing human fetal dorsal root ganglia and spinal cord. The technique used for examination of cryosections was double-label fluorescence with monoclonal antibodies. Both proteins were present in the nerve fibres inside the ganglia of 6- and 8-week-old embryos. During further development the expression of vimentin continued to increase in the satellite cells, but was found to be decreasing in the ganglion cells. Phosphorylated NF-H was found in the processes of ganglion cells, as well as in the perikarya at all developmental stages. In the spinal cord of 6- and 8-week-old embryos, phosphorylated NF-H protein was found in the longitudinal fibres of the marginal layer and in processes of the mantle zone; some of the fibres also contained vimentin. Later the co-expression of the two proteins ceased and vimentin was found only in glial and mesenchymal derivatives. Phosphorylated NF-H was located, at all developmental stages, in the axons of both white and grey matter, but not in the neuronal perikarya. The results indicate that phosphorylation of the NF-H in human dorsal root ganglia starts in the perikarya of the ganglion cells while in the ganglion cells of the spinal cord it takes place in the axons.

• MeSH
• Phosphorylation MeSH
• Gestational Age MeSH
• Humans MeSH
• Spinal Cord embryology   metabolism   MeSH
• Molecular Weight MeSH
• Antibodies, Monoclonal MeSH
• Neurofilament Proteins chemistry   immunology   metabolism   MeSH
• Intermediate Filament Proteins metabolism   MeSH
• Antibody Specificity MeSH
• Ganglia, Spinal embryology   metabolism   MeSH
• Vimentin metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antibodies, Monoclonal MeSH
• neurofilament protein H MeSH Browser
• Neurofilament Proteins MeSH
• Intermediate Filament Proteins MeSH
• Vimentin MeSH

Individual beta-tubulin isoforms in developing mouse brain were characterized using immunoblotting, after preceding high-resolution isoelectric focusing, with monoclonal antibodies against different structural regions of beta-tubulin. Some of the antibodies reacted with a limited number of tubulin isoforms in all stages of brain development and in HeLa cells. The epitope for the TU-14 antibody was located in the isotype-defining domain and was present on the beta-tubulin isotypes of classes I, II and IV, but absent on the neuron-specific class-III isotype. The data suggest that non-class-III beta-tubulins in mouse brain are substrates for developmentally regulated post-translational modifications and that beta-tubulins of non-neuronal cells are also post-translationally modified.

• MeSH
• Epitopes analysis   MeSH
• HeLa Cells MeSH
• Immunoblotting MeSH
• Isoelectric Focusing MeSH
• Isomerism MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Antibodies, Monoclonal MeSH
• Brain growth & development   immunology   metabolism   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Protein Processing, Post-Translational physiology   MeSH
• Amino Acid Sequence MeSH
• Tubulin immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Epitopes MeSH
• Antibodies, Monoclonal MeSH
• Tubulin MeSH