Soluble tubulin complexes, gamma-tubulin, and their changing distribution in the zebrafish (Danio rerio) ovary, oocyte and embryo

Tubulin dynamics, i.e., the interchange of polymeric and soluble forms, is important for microtubule (MTs) cellular functions, and thus plays essential roles in zebrafish oogenesis and embryogenesis. A novel finding in this study revealed that there were soluble pools of tubulins in zebrafish oocytes that were sequestered and maintained in a temporary "oligomeric" state, which retained assembling and disassembling potential (suggested by undetected acetylated tubulin, marker of stable tubulin), but lacked abilities to assemble into MTs spontaneously in vivo. Using differential centrifugation, gel chromatography and DM1A-probed western blot, soluble alpha-tubulin was found to be associated with large molecular weight complexes (MW range to over 2 MDa) which were reduced in amount by the blastula stage, especially in some batches of embryos, with a concomitant decrease in soluble tubulin. Complexes (MW range less than 2 MDa) then increased in the gastrula with an increase in soluble alpha-tubulin. Two different anti-gamma-tubulin monoclonal antibodies, GTU 88 and TU 30, revealed the existence of soluble gamma-tubulin in both zebrafish oocytes and embryos, which also decreased by the blastula stage and increased in the gastrula stage. Soluble alpha-tubulin and gamma-tubulin extracted from zebrafish ovaries, oocytes and embryos co-localized in fractions on three different columns: S-200 Sephacryl, DEAE and Superose-6b. The soluble tubulin complexes were competent to assemble into MTs in vitro induced by taxol, and gamma-tubulin was co-localized with assembled MTs. These soluble tubulin complexes were stable during freeze-thaw cycles and resisted high ionic interaction (up to 1.5 M NaCl). Furthermore, some ovarian soluble alpha-tubulin could be co-immunoprecipitated with gamma-tubulin, and vice versa. Two antibodies specific for Xenopus gamma-tubulin ring complex proteins (Xgrip 109 and Xgrip 195) detected single bands from ovarian extracts in western blots, suggesting the existence of Xgrip 109 and Xgrip 195 homologues in zebrafish. These findings, together with recent work on gamma-tubulin ring complexes in oocytes, eggs and embryos of other species, suggest that soluble gamma-tubulin-associated protein complexes may be involved in regulating tubulin dynamics during zebrafish oogenesis and embryogenesis.     

Soluble tubulin complexes in oocytes of the common leopard frog, Rana pipiens, contain gamma-tubulin

Oocytes of the leopard frog, Rana pipiens, contain soluble tubulin which was previously shown to exist predominantly in megadalton (MDa) fractions and that fails to readily assemble in vitro. In order to further characterize these tubulin complexes, DEAE Sepharose chromatography, Sephacryl S-300 size exclusion columns and specific immunoprecipitation were used. The results revealed the presence of alpha-, beta-, and gamma-tubulin associated with several other proteins in the soluble fraction of Rana pipiens ovarian oocytes. These Rana oocyte tubulin complexes appear to be analogous to those recently reported in Xenopus ovulated eggs as gamma-tubulin ring complexes. This seems true since both size (estimates, i.e. approximately 2MDa) and protein components are similar. Furthermore, both alpha- and gamma-tubulin antibodies immunoprecipitated identical protein bands from Rana oocyte soluble fraction. These putative Rana gamma-tubulin ring proteins include 107, 97, 95, 90 and 75 kDa components which are similar in size to those found in Xenopus and other species. Rana appears to belong to a select group in which gamma-tubulin complexes contain significant alpha- and beta-tubulin (i.e., Xenopus and sheep), while other species such as Drosophila, Aspergillus, Saccharomyces, human cells and many other mammalian cells tested lack the other tubulin components. The heterogeneity in both size and protein components of Rana oocyte gamma-tubulin ring complexes may reflect different states of tubulin complex assembly. The lower vertebrate oocyte is hypothesized to act as a repository and prestaging point for the assembly of gamma-tubulin ring complexes which will become the maternal contribution to the centrosomes of the embryo. While the gamma-tubulin ring complexes of vertebrate eggs have been described previously, this is the first report biochemically characterizing soluble gamma-tubulin complexes in vertebrate ovarian oocytes.     

Complexes of gamma-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that gamma-tubulin (gamma-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, gamma-tubulin, and with anti-phosphotyrosine antibody revealed that gamma-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated gamma-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing gamma-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of gamma-tubulin interaction with tubulin dimers or other proteins during neurogenesis.     

Tubulin binding sites on gamma-tubulin: identification and molecular characterization

gamma-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that gamma-tubulin interacts with tubulin to accomplish its cellular functions. However, such an interaction has been difficult to demonstrate and to characterize at the molecular level. gamma-Tubulin is a poorly soluble protein, not amenable to biochemical studies in a purified form as yet. Therefore basic questions concerning the existence and properties of tubulin binding sites on gamma-tubulin have been difficult to address. Here we have performed a systematic search for tubulin binding sites on gamma-tubulin using the SPOT peptide technique. We find a specific interaction of tubulin with six distinct domains on gamma-tubulin. These domains are clustered in the central part of the gamma-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulin binding domains of gamma-tubulin bind with nanomolar K(d)s to tubulin dimers. These peptides do not interfere measurably with microtubule assembly in vitro and associate with microtubules along the polymer length. On the tertiary structure, the gamma-tubulin peptides cluster to surface regions on both sides of the molecule. Using SPOT analysis, we also find peptides interacting with gamma-tubulin in both the alpha- and beta-tubulin subunits. The tubulin peptides cluster to surface regions on both sides of the alpha- and beta- subunits. These data establish gamma-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that gamma-tubulin and tubulin dimers associate through lateral interactions.     

Gamma-tubulins and their functions

gamma-Tubulin is a ubiquitous phylogenetically conserved member of tubulin superfamily. In comparison with alpha beta-tubulin dimers, it is a low abundance protein present within the cells in both various types of microtubule-organizing centers and cytoplasmic protein complexes. gamma-Tubulin small complexes are subunits of the gamma-tubulin ring complex, which is involved in microtubule nucleation and capping of the minus ends of microtubules. In the past years important findings have advanced the understanding of the structure and function of gamma-tubulin ring complexes. Recent evidences suggest that the functions of gamma-tubulin extend beyond microtubule nucleation.     

Chaperonin-mediated folding of vertebrate actin-related protein and gamma-tubulin

The folding of actin and tubulin is mediated via interaction with a heteromeric toroidal complex (cytoplasmic chaperonin) that hydrolyzes ATP as part of the reaction whereby native proteins are ultimately released. Vertebrate actin-related protein (actin-RPV) (also termed centractin) and gamma-tubulin are two proteins that are distantly related to actin and tubulin, respectively: gamma-tubulin is exclusively located at the centrosome, while actin-RPV is conspicuously abundant at the same site. Here we show that actin-RPV and gamma- tubulin are both folded via interaction with the same chaperonin that mediates the folding of beta-actin and alpha- and beta-tubulin. In each case, the unfolded polypeptide forms a binary complex with cytoplasmic chaperonin and is released as a soluble, monomeric protein in the presence of Mg-ATP and the presence or absence of Mg-GTP. In contrast to alpha- and beta-tubulin, the folding of gamma-tubulin does not require the presence of cofactors in addition to chaperonin itself. Monomeric actin-RPV produced in in vitro folding reactions cocycles efficiently with native brain actin, while in vitro folded gamma- tubulin binds to polymerized microtubules in a manner consistent with interaction with microtubule ends. Both monomeric actin-RPV and gamma- tubulin bind to columns of immobilized nucleotide: monomeric actin-RPV has no marked preference for ATP or GTP, while gamma-tubulin shows some preference for GTP binding. We show that actin-RPV and gamma-tubulin compete with one another, and with beta-actin or alpha-tubulin, for binary complex formation with cytoplasmic chaperonin.     

Microtubule nucleation

Microtubule nucleation is the process in which several tubulin molecules interact to form a microtubule seed. Microtubule nucleation occurs spontaneously in purified tubulin solutions, and molecular intermediates between tubulin dimers and microtubules have been identified. Microtubule nucleation is enhanced in tubulin solutions by the addition of gamma-tubulin or various gamma-tubulin complexes. In vivo, microtubule assembly is usually seeded by gamma-tubulin ring complexes. Recent studies suggest, however, that microtubule nucleation can occur in the absence of gamma-tubulin, and that gamma-tubulin may have other cell functions apart from being a major component of the gamma-tubulin ring complex.     

Cloning and expression of the tubulin genes in barley

Tubulins are encoded by small gene families in plants. Based on the barley EST collection, cDNAs for alpha-, beta-, and gamma-tubulins were selected. Five genes for alpha-tubulin, eight newly identified beta-tubulin sequences and one gamma-tubulin gene were found to be expressed in barley. In silico analysis of relative abundance of the distinct tubulin sequences among ESTs derived from different libraries revealed that the various tubulin genes differed in their level of expression, and to some extent were tissue specific.     

Class III β-Tubulin and γ-Tubulin are Co-expressed and Form Complexes in Human Glioblastoma Cells

We have previously shown that the neuronal-associated class III beta-tubulin isotype and the centrosome-associated gamma-tubulin are aberrantly expressed in astrocytic gliomas (Cell Motil Cytoskeleton 2003, 55:77-96; J Neuropathol Exp Neurol 2006, 65:455-467). Here we determined the expression, distribution and interaction of betaIII-tubulin and gamma-tubulin in diffuse-type astrocytic gliomas (grades II-IV) (n = 17) and the human glioblastoma cell line T98G. By immunohistochemistry and immunofluorescence microscopy, betaIII-tubulin and gamma-tubulin were co-distributed in anaplastic astrocytomas and glioblastomas and to a lesser extent, in low-grade diffuse astrocytomas (P < 0.05). In T98G glioblastoma cells betaIII-tubulin was associated with microtubules whereas gamma-tubulin exhibited striking diffuse cytoplasmic staining in addition to its expectant centrosome-associated pericentriolar distribution. Treatment with different anti-microtubule drugs revealed that betaIII-tubulin was not associated with insoluble gamma-tubulin aggregates. On the other hand, immunoprecipitation experiments unveiled that both tubulins formed complexes in soluble cytoplasmic pools, where substantial amounts of these proteins were located. We suggest that aberrant expression and interactions of betaIII-tubulin and gamma-tubulin may be linked to malignant changes in glial cells.     

gamma-Tubulin in Leishmania: cell cycle-dependent changes in subcellular localization and heterogeneity of its isoforms

A panel of six anti-peptide antibodies recognizing epitopes in different regions of the gamma-tubulin molecule was used for the characterization and localization of gamma-tubulin during cell cycle in Leishmania promastigotes. Immunofluorescence microscopy revealed the presence of gamma-tubulin in the basal bodies, posterior pole of the cell, and in the flagellum. Furthermore, the antibodies showed punctuate staining in the subpellicular microtubule. This complex localization pattern was observed in both interphase and dividing cells, where staining of posterior poles and the subpellicular corset was more prominent. In posterior poles, gamma-tubulin co-distributed with the 210-kDa microtubule-interacting protein and the 57-kDa protein immunodetected with anti-vimentin antibody. Immunogold electron microscopy on thin sections of isolated flagella showed that gamma-tubulin was associated with the paraflagellar rod (PFR) that runs adjacent to the axonemal microtubules. Under different extraction conditions, gamma-tubulin in Leishmania was found only in insoluble cytoskeletal fractions, in contrast to tubulin dimers that were both in soluble and cytoskeletal pool. Two-dimensional electrophoresis revealed multiple charge variants of gamma-tubulin. Posttranslational modifications of Leishmania gamma-tubulin might therefore have an important role in the regulation of microtubule nucleation and interaction with other proteins. The complex pattern of gamma-tubulin localization and its properties indicate that gamma-tubulin in Leishmania might have other function(s) besides microtubule nucleation.     

Tetrahymena thermophila contains a conventional gamma-tubulin that is differentially required for the maintenance of different microtubule-organizing centers

The gene (GTU1) encoding Tetrahymena thermophila gamma-tubulin was cloned and analyzed. GTU1 is a single-copy, essential gene encoding a conventional gamma-tubulin. HA-tagged GTU1p localizes to four microtubule-organizing centers (MTOCs) in vegetative cells: basal bodies (BBs), macronuclear envelopes, micronuclear envelopes, and contractile vacuole pores. gamma-Tubulin function was studied by placing the GTU1 gene under control of an inducible-repressible promoter. Overexpression of GTU1 had no detectable effect on cell growth or morphology. Depletion of gamma-tubulin resulted in marked changes in cell morphology and in MT bundling. MTOCs showed different sensitivities to gamma-tubulin depletion, with BBs being the most sensitive. gamma-Tubulin was required not only for the formation of new BBs but also for maintenance of mature BBs. BBs disappeared in stages, first losing gamma-tubulin and then centrin and glutamylated tubulin. When GTU1 expression was reinduced in depleted cells, BBs reformed rapidly, and the normal, highly organized structure of the Tetrahymena cell cortex was reestablished, indicating that the precise patterning of the cortex can be formed de novo.     

Gamma-tubulin complexes and microtubule organization

Microtubule nucleation requires gamma-tubulin, which exists in two main protein complexes: the gamma-tubulin small complex, and the gamma-tubulin ring complex. During mitosis, these complexes accumulate at the centrosome to support spindle formation. Gamma-tubulin complexes are also present at non-centrosomal microtubule nucleation sites, both in interphase and in mitosis. In interphase, non-centrosomal nucleation enables the formation of microtubule bundles or networks of branched microtubules. Gamma-tubulin complexes may be involved not only in microtubule nucleation, but also in regulating microtubule dynamics. Recent findings indicate that the dynamics of microtubule plus-ends are altered, depending on the expression of gamma-tubulin complex proteins.     

The expression of gelatinase A (MMP-2) is required for normal development of zebrafish embryos

Gelatinase A, also called matrix metalloproteinase 2 (MMP-2), belongs to the matrix metalloproteinase (MMP) family. MMP-2 cleaves type IV collagen, denatured collagen (gelatin), and other extracellular matrix (ECM) components. MMP-2 has been reported to be involved in a number of biological and pathological processes, but previous studies have not indicated that its expression is essential for early embryogenesis. In the current study, we have utilized zebrafish as a developmental model to study the role of MMP-2 during embryogenesis. We have successfully isolated a zebrafish MMP-2 (zMMP-2) homologue showing over 80% identity and over 90% similarity to its human counterpart. In situ analysis showed that zMMP-2 was expressed as early as the one-cell stage implying a maternal origin during oogenesis, and embryos continued to express zMMP-2 through at least the 72-h stage of development. RT-PCR analysis confirmed the in situ expression pattern and gelatin zymography indicated that a metalloproteinase with the same gel mobility as vertebrate MMP-2 was present in zebrafish embryos. Injection of zMMP-2 antisense morpholino oligonucleotides into 1- to 4-cell embryos resulted in a truncated axis, monitored through 72 h of development indicating that this metalloproteinase plays an important role in zebrafish embryogenesis. Monpholino-induced alterations in development began to be observed at 12 h of embryogenesis based on morphological and axis marker studies. The results obtained in zebrafish are in contrast to murine knockout studies that indicate that MMP-2 does not have a major role in mouse embryogenesis.Edited by D. Tautz     

Identification and isolation of a BTB-POZ-containing gene expressed in oocytes and early embryos of the zebrafish Danio rerio.

In this report, we describe the cloning of a cDNA from the zebrafish Danio rerio encoding a protein containing a BTB-POZ domain closely resembling the BTBD1 and BTBD2 proteins previously identified in mammals. However, unlike other BTB-POZ-containing genes, expression of this gene in adults is most abundant in oocytes, where the RNA can be detected at all stages of oogenesis examined. The presence of the RNA persists through early cleavage, but is decreased significantly by gastrulation. Although the function of this gene has yet to be determined, its resemblance to the BTB-POZ family of genes coupled with its expression pattern suggests that it may have an important function in oogenesis and (or) early zebrafish development.     

Characterization of the poly(A) binding proteins expressed during oogenesis and early development of Xenopus laevis

During vertebrate oogenesis and early embryogenesis, gene expression is governed mainly by translational control. The recruitment of Poly(A) Binding Protein (PABP) during poly(A) tail lengthening appears to be the key to translational activation during this period of development in Xenopus laevis. We showed that PABP1 and ePABP proteins are both present during oogenesis and early development. We selected ePABP as an eRF3 binding protein in a two-hybrid screening of a X. laevis cDNA library and demonstrated that this protein is associated with translational complexes. It can complement essential functions of the yeast homologue Pab1p. We discuss specific expression patterns of the finely tuned PABP1 and ePABP proteins.