The tektin family of microtubule-stabilizing proteins

Tektins are insoluble alpha-helical proteins essential for the construction of cilia and flagella and are found throughout the eukaryotes apart from higher plants. Being almost universal but still fairly free to mutate, their coding sequences have proved useful for estimating the evolutionary relationships between closely related species. Their protein molecular structure, typically consisting of four coiled-coil rod segments connected by linkers, resembles that of intermediate filament (IF) proteins and lamins. Tektins assemble into continuous rods 2 nm in diameter that are probably equivalent to subfilaments of the 10 nm diameter IFs. Tektin and IF rod sequences both have a repeating pattern of charged amino acids superimposed on the seven-amino-acid hydrophobic pattern of coiled-coil proteins. The length of the repeat segment matches that of tubulin subunits, suggesting that tektins and tubulins may have coevolved, and that lamins and IFs may have emerged later as modified forms of tektin. Unlike IFs, tektin sequences include one copy of a conserved peptide of nine amino acids that may bind tubulin. The 2 nm filaments associate closely with tubulin in doublet and triplet microtubules of axonemes and centrioles, respectively, and help to stabilize these structures. Their supply restricts the assembled lengths of cilia and flagella. In doublet microtubules, the 2 nm filaments may also help to organize the longitudinal spacing of accessory structures, such as groups of inner dynein arms and radial spokes.     

Tektin interactions and a model for molecular functions

Tektins from echinoderm flagella were analyzed for microheterogeneity, self-associations and association with tubulin, resulting in a general model of tektin filament structure and function applicable to most eukaryotic cilia and flagella. Using a new antibody to tektin consensus peptide RPNVELCRD, well-characterized chain-specific antibodies and quantitative gel densitometry, tektins A, B and C were found to be present in equimolar amounts in Sarkosyl-urea-stable filaments. In addition, two isoforms of tektin A are present in half-molar ratios to tektins B and C. Cross-linking of AB filaments indicates in situ nearest neighbor associations of tektin A1B and A2B heterodimers, -trimers, -tetramers and higher oligomers. Soluble purified tektin C is cross-linked as homodimers, trimers and tetramers, but not higher oligomers. Tektin filaments associate with both loosely bound and tightly bound tubulin, and with the latter in a 1:1 molar ratio, implying a specific, periodic association of tightly bound tubulin along the tektin axis. Similarly, in tektin-containing Sarkosyl-stable protofilament ribbons, two polypeptides ( approximately 67/73 kDa, homologues of rib72, efhc1 and efhc2) are present in equimolar ratios to each other and to individual tektins, co-fractionating with loosely bound tubulin. These results suggest a super-coiled arrangement of tektin filaments, the organization of which has important implications for the evolution, assembly and functions of cilia and flagella.     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

Localization of tektin filaments in microtubules of sea urchin sperm flagella by immunoelectron microscopy

Extraction of doublet microtubules from the sperm flagella of the sea urchin Strongylocentrotus purpuratus with sarkosyl (0.5%)-urea (2.5 M) yields a highly pure preparation of "tektin" filaments that we have previously shown to resemble intermediate filament proteins. They form filaments 2-3 nm in diameter as seen by negative stain electron microscopy and are composed of approximately equal amounts of three polypeptide bands with apparent molecular weights of 47,000, 51,000, and 55,000, as determined by SDS PAGE. We prepared antibodies to this set of proteins to localize them in the doublet microtubules of S. purpuratus and other species. Tektins and tubulin were antigenically distinct when tested by immunoblotting with affinity-purified antitektin and antitubulin antibodies. Fixed sperm or axonemes from several different species of sea urchin showed immunofluorescent staining with antitektin antibodies. We also used antibodies coupled to gold spheres to localize the proteins by electron microscopy. Whereas a monoclonal antitubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol. 93:576-582) decorates intact microtubules along their lengths, antitektins labeled only the ends of intact microtubules and sarkosyl-insoluble ribbons. However, if microtubules and ribbons attached to electron microscope grids were first extracted with sarkosyl-urea, the tektin filaments that remain were decorated by antitektin antibodies throughout their length. These results suggest that tektins form integral filaments of flagellar microtubule walls, whose antigenic sites are normally masked, perhaps by the presence of tubulin around them.     

The spatial and temporal expression of Tekt1, a mouse tektin C homologue, during spermatogenesis suggest that it is involved in the development of the sperm tail basal body and axoneme

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. Recently we described the sequence of the first mammalian tektin protein, Tekt1 (from mouse testis), which is most homologous with sea urchin tektin C. We have now investigated the temporal and spatial expression of Tekt1 during mouse male germ cell development. By in situ hybridization analysis TEKT1 RNA expression is detected in spermatocytes and in round spermatids in the mouse testis. Immunofluorescence microscopy analysis with anti-Tekt1 antibodies showed no distinct labeling of any subcellular structure in spermatocytes, whereas in round spermatids anti-Tekt1 antibodies co-localize with anti-ANA antibodies to the centrosome. At a later stage, elongating spermatids display a larger area of anti-Tektl staining at their caudal ends; as spermiogenesis proceeds, the anti-Tekt1 staining disappears. Together with other evidence, these results provide the first intraspecies evidence that Tekt1 is transiently associated with the centrosome, and indicates that Tekt1 is one of several tektins to participate in the nucleation of the flagellar axoneme of mature spermatozoa, perhaps being required to assemble the basal body.     

Tektin 3 is required for progressive sperm motility in mice

Tektins are evolutionarily conserved flagellar (and ciliary) filamentous proteins present in the axoneme and peri-axonemal structures in diverse metazoan species. We have previously shown that tektin 3 (TEKT3) and tektin 4 (TEKT4) are male germ cell-enriched proteins, and that TEKT4 is essential for coordinated and progressive sperm motility in mice. Here we report that male mice null for TEKT3 produce sperm with reduced motility (47.2% motility) and forward progression, and increased flagellar structural bending defects. Male TEKT3-null mice however maintain normal fertility in two different genetic backgrounds tested, in contrast to TEKT4-null mice. Furthermore, male mice null for both TEKT3 and TEKT4 show subfertility on a mixed B6;129 genetic background, significantly different from either single knockouts, suggesting partial nonredundant roles for these two proteins in sperm physiology. Our results suggest that tektins are potential candidate genes for nonsyndromic asthenozoospermia in humans.     

Equimolar heterodimers in microtubules

Two equimolar beta chains can be resolved from sea urchin sperm flagellar and scallop gill ciliary tubulins, and from certain brain tubulins as well, using the Triton X-100-acid-urea polyacrylamide gel system commonly used for histone analysis. The beta chains are identified as such from their mobility on urea-free SDS PAGE, from amino acid composition, and from tryptic peptide distribution. Scallop beta chains have almost identical amino acid profiles but they differ by one tryptic peptide. Optimal conditions for beta chain resolution are very species-dependent, with some closely related species showing either maximal or no beta chain separation. In addition, beef brain tubulin on Triton X-100-acid-urea electrophoresis and scallop gill ciliary tubulin upon isoelectric focusing in the presence of SDS show two approximately equimolar alpha chains. These data, indicating equimolar amounts of two potentially different tubulin heterodimers from a variety of microtubule types, support a model for microtubule structure wherein protofilaments consist of alternating heterodimers of two kinds, generating a 16-nm (2-dimer) axial repeat.     

Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.     

Relationship between tektins and intermediate filament proteins: an immunological study

Affinity-purified antibodies raised against three flagellar tektins (tektin A, B, and C) from each of two sea urchin species (Lytechinus pictus and Strongylocentrotus purpuratus) were used to study the immunological relationship between tektins and intermediate filament proteins. By immunofluorescence microscopy, several antitektins revealed a staining of intermediate filament-like arrays in three vertebrate cell lines tested. Immunoelectron microscopy substantiated the cross reaction of antitektins with intermediate filaments. When the cells were treated with cytochalasin B, the arrangement of the filaments recognized by anti-(Lp)-tektin B was altered; the alteration observed is typical for keratin filaments. By immunoblot, it was found that anti-(Lp)-tektin B cross reacted with two isoforms or different proteins of approximately 54 kD with pIs of 6.1 and 6.2 in human carcinoma epithelia (HeLa) cells and with two isoforms or different proteins of approximately 55 kD with pIs of 6.1 and 6.3 in pig kidney epithelia (LLC-PK1) cells. Furthermore, when antitektin antibodies were affinity purified with the 54 kD HeLa keratin, these keratin-specific antibodies again restained the original tektins on immunoblots. From these observations, it can be concluded that tektins and keratins are to a certain extent immunologically related. To determine the degree of the immunological relationship, tektin filaments and purified intermediate filaments from HeLa cells were cleaved with alpha-chymotrypsin and examined by quantitative immunoblot analysis. On immunoblots of digested tektins from L. pictus, anti-(Lp)-tektin B recognized several cleavage products in the range of 20 kD to 46 kD. However, when immunoblots of digested intermediate filaments from HeLa cells were probed, the cross reaction of anti-(Lp)-tektin B with HeLa keratins was eliminated by more than 98% within 2 min, suggesting that tektins have epitopes in common with the end domains of certain keratins.     

Antarctic fish tubulins: heterogeneity, structure, amino acid compositions and charge

1. Tubulins purified from the brain tissues of three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus) contain equimolar quantities of the alpha and beta chains and are free of microtubule-associated proteins (MAPs) and other non-tubulin proteins. 2. When examined by isoelectric focusing and by two-dimensional electrophoresis, brain tubulins from the Antarctic fishes were found to be highly heterogeneous; each was resolved into 15-20 distinct variants. The range of isoelectric points displayed by the Antarctic fish tubulins (5.30-5.75) is slightly more basic than that of bovine brain tubulin (5.25-5.60). 3. Peptide mapping demonstrated that tubulins from the Antarctic fishes and the cow differ in structure. 4. The amino acid compositions of piscine and mammalian tubulins are similar, but the Antarctic fish tubulins apparently contain fewer glutamyl and/or glutaminyl residues than do tubulins from the temperate channel catfish (Ictalurus punctatus) and the cow. 5. Native tubulin from N. coriiceps neglecta possesses 1-2 fewer net negative charges per tubulin dimer than does bovine tubulin. 6. We suggest that the enhanced assembly of Antarctic fish tubulins at low temperatures (-2 to +2 degrees C) results from adaptive, perhaps subtle, changes in their tubulin subunits.     

Changes in the polypeptide patterns of barley seedlings exposed to jasmonic Acid and salinity

Soluble and thylakoid membrane proteins of jasmonic acid (JA)-treated and salt-stressed barley (Hordeum vulgare L.) seedlings were investigated using 15% sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. High JA concentrations induced marked quantitative and qualitative changes in polypeptide profiles concerning mainly the proteins with approximately equal mobility, as in NaCl-stressed plants. The most obvious increase in thylakoid polypeptide band intensity was at 55 to 57 kilodaltons (kD). The relative share of some polypeptides with apparent molecular masses above 66 kD and of polypeptides with lower molecular masses in the region of 20.5 to 15 kD was enhanced. At the same time, one new band at 31 to 31.5 kD was well expressed at 25 and 250 micromolar JA concentrations and became discernible in the 100 micromolar NaCl-treated plants. The intensity of some polypeptides of soluble proteins (molecular masses of 60, 47, 37, 30, and 23.4 kD) increased with increasing JA concentration, whereas the intensities of other polypeptide bands (55, 21.4, and 15 kD) decreased. Enhanced levels of 60-, 47-, 34-, and 30-kD polypeptides and reduced levels of 55- and 15-kD polypeptides were present in NaCl-treated plants. The appearance of one new polypeptide, of 25.1 kD, was observed only in NaCl-treated plants. At 100 millimolar NaCl, an eightfold increase in proline content was observed while at 250 micromolar JA, the proline content was threefold over the control. It is hypothesized that exogenously applied jasmonates act as stress agents. As such, they provoke alterations in the proline content and they can modulate typical stress responses by induction of stress proteins.     

Molecular cloning of haploid germ cell-specific tektin cDNA and analysis of the protein in mouse testis.

Tektins are a class of proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules. We report here the molecular cloning of a new member of the tektin family, tektin-t, identified from a mouse haploid germ cell-specific cDNA library. Tektin-t mRNA encodes a protein of 430 deduced amino acids possessing RSNVELCRD, the conserved sequence of tektin family proteins. Western blotting showed a single band having a molecular weight of 86 kDa in the mouse testis. Immunohistochemistry of the testis showed that tektin-t is localized in the flagella of elongating spermatids from developmental step 15 to maturity.     

Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.     

Cloning, purification, and polymerization of Capsicum annuum recombinant alpha and beta tubulin

Alpha and beta tubulin genes were cloned from the Capsicum annuum leaves using rapid amplification of cDNA ends (RACE)-PCR. Nucleotide sequence analysis revealed that 1,353 bp Capsicum annuum alpha/beta-tubulin (CAnm alpha/beta-TUB) encodes a protein of 450 amino acids (aa) each. The recombinant alpha/beta tubulin was overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with 0.2 mM isopropyl-beta-D-thiogalactopyranoside (IPTG), and its content was as high as 50% of the total protein content. Effective fusion protein purification and refolding are described. The average yields of alpha and beta tubulin were 2.0 and 1.3 mg/l of culture respectively. The apparent molecular weight of each tubulin was estimated to be 55 kDa by SDS-polyacrylamide gel electrophoresis (PAGE). The tubulin monomers were found to be assembly competent using a standard dimerization assay, and also retained antigenicity with anti-His/T7 antibodies. The purified tubulins were polymerized to microtubule-like structures in the presence of 2 mM guanosine 5'-triphosphate (GTP).     

Structural and biological characterization of the tubulin interaction with dinitroanilines

Characteristics of the interaction of dinitroaniline compounds with tubulin molecules have an extremely high selectivity: these substances efficiently bind to the tubulins of both plant and protozoan origins and practically do not interact with any animal and fungal tubulins despite a very high similarity between the corresponding sequences. This work summarizes and comprehensively analyzes the specific structural features and mechanisms of these interactions, in particular, the patterns of the structure and arrangement of dinitroaniline binding sites on the surface of different tubulin subunits and tubulins of various origins. Dinitroaniline binding sites are localized to the surface of longitudinal contacts between tubulin subunits and contain diamine amino acid residues (lysine or arginine), which bind the nitrile group of dinitroanilines. The localizations of these sites on the surface of identical subunits of different origins (for example, α-tubulins of plants and protozoans) coincide; however, the location of these binding sites on the surfaces of tubulin α- and β-subunits is different. The characterized sites can also be potential binding sites for other antimicrotubule compounds, in particular, cyanoacrylates.     

Cold-adapted tubulins in the glacier ice worm, Mesenchytraeus solifugus

Glacier ice worms, Mesenchytraeus solifugus and related species, are the only known annelids that survive obligately in glacier ice and snow. One fundamental component of cold temperature adaptation is the ability to polymerize tubulin, which typically depolymerizes at low physiological temperatures (e.g., <10 degrees C) in most temperate species. In this study, we isolated two alpha-tubulin (Msalpha1, Msalpha2) and two beta-tubulin (Msbeta1, Msbeta2) subunits from an ice worm cDNA library, and compared their predicted amino acid sequences with homologues from other cold-adapted organisms (e.g., Antarctic fish, ciliate) in an effort to identify species-specific amino acid substitutions that contribute to cold temperature-dependent tubulin polymerization. Our comparisons and predicted protein structures suggest that ice worm-specific amino acid substitutions stabilize lateral contact associations, particularly between beta-tubulin protofilaments, but these substitutions occur at different positions in comparison with other cold-adapted tubulins. The ice worm tubulin gene family appears relatively small, comprising one primary alpha- and one primary beta-tubulin monomers, though minor isoforms and psuedogenes were identified. Our analyses suggest that variation occurs in the strategies (i.e., species-specific amino acid substitutions, gene number) by which cold-adapted taxa have evolved the ability to polymerize tubulin at low physiological temperatures.     

Posttranslational modification of brain tubulins from the Antarctic fish Notothenia coriiceps: reduced C-terminal glutamylation correlates with efficient microtubule assembly at low temperature

We have shown previously that the tubulins of Antarctic fish assemble into microtubules efficiently at low temperatures (-2 to +2 degrees C) due to adaptations intrinsic to the tubulin subunits. To determine whether changes in posttranslational glutamylation of the fish tubulins may contribute to cold adaptation of microtubule assembly, we have characterized C-terminal peptides from alpha- and beta-tubulin chains from brains of adult specimens of the Antarctic rockcod Notothenia coriiceps by MALDI-TOF mass spectrometry and by Edman degradation amino acid sequencing. Of the four fish beta-tubulin isotypes, nonglutamylated isoforms were more abundant than glutamylated isoforms. In addition, maximal glutamyl side-chain length was shorter than that observed for mammalian brain beta tubulins. For the nine fish alpha-tubulin isotypes, nonglutamylated isoforms were also generally more abundant than glutamylated isoforms. When glutamylated, however, the maximal side-chain lengths of the fish alpha tubulins were generally longer than those of adult rat brain alpha chains. Thus, Antarctic fish adult brain tubulins are glutamylated differently than mammalian brain tubulins, resulting in a more heterogeneous population of alpha isoforms and a reduction in the number of beta isoforms. By contrast, neonatal rat brain tubulin possesses low levels of glutamylation that are similar to that of the adult fish brain tubulins. We suggest that unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.     

Isolation and partial characterization of a 110-kD dimer actin-binding protein

Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I.