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: binding to the centrosome, regulation and microtubule nucleation

Microtubule assembly is initiated in vivo by gamma-tubulin complexes. Cytoplasmic gamma-tubulin complexes are targeted to centrosomes or to other microtubule organizing centers (MTOCs) via a set of so called gamma-tubulin complex binding proteins (GTBPs) that probably interact with the conserved Spc97p/Spc98p protein family of gamma-tubulin complexes. In other cell types, gamma-tubulin complexes may initiate microtubule formation near chromosomes in a MTOC-independent manner. Recently, major advances have been achieved through the finding that gamma-tubulin, Spc97p and Spc98p form a conserved core that is probably responsible for microtubule nucleation, and by the discovery that a yeast GTBP is regulated in a cell-cycle-dependent manner and in response to an external signal. Furthermore, it was found that the small GTPase Ran in its GDP-bound state may promote spindle assembly. In addition, an essential function of gamma-tubulin in basal body duplication has been demonstrated in Paramecium.     

Behavior of delta-tubulin during spindle formation in Xenopus oocytes: requirement of cytoplasmic dynein-dependent translocation

Vertebrate oocytes do not contain centrosomes and therefore form an acentrosomal spindle during oocyte maturation. gamma-Tubulin is known to be essential for nucleation of microtubules at centrosomes, but little is known about the behaviour and role of gamma-tubulin during spindle formation in oocytes. We first observed sequential localization of gamma-tubulin during spindle formation in Xenopus oocytes. gamma-Tubulin assembled in the basal regions of the germinal vesicle (GV) at the onset of germinal vesicle breakdown (GVBD) and remained on the microtubule-organizing centre (MTOC) until a complex of the MTOC and transient-microtubule array (TMA) reached the oocyte surface. Prior to bipolar spindle formation, oocytes formed an aggregation of microtubules and gamma-tubulin was concentrated at the centre of the aggregation. At the late stage of bipolar spindle formation, gamma-tubulin accumulated at each pole. Anti-dynein antibody disrupted the localization of gamma-tubulin, indicating that the translocation described above is dependent on dynein activity. We finally revealed that XMAP215, a microtubule-associated protein cooperating with gamma-tubulin for the assembly of microtubules, but not gamma-tubulin, was phosphorylated during oocyte maturation. These results suggest that gamma-tubulin is translocated by dynein to regulate microtubule organization leading to spindle formation and that modification of the molecules that cooperate with gamma-tubulin, but not gamma-tubulin itself, is important for microtubule reorganization.     

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.     

Human 76p: A new member of the gamma-tubulin-associated protein family

The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.     

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.     

Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.     

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.     

Regulation of microtubule formation in activated mast cells by complexes of gamma-tubulin with Fyn and Syk kinases

Aggregation of the high-affinity IgE receptors (FcepsilonRIs) 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 FcepsilonRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcepsilonRI 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 gamma-tubulin. Immunoprecipitation experiments revealed that gamma-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 gamma-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 gamma-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 gamma-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.     

Regulation of microtubule nucleation from membranes by complexes of membrane-bound gamma-tubulin with Fyn kinase and phosphoinositide 3-kinase

The molecular mechanisms controlling microtubule formation in cells with non-centrosomal microtubular arrays are not yet fully understood. The key component of microtubule nucleation is gamma-tubulin. Although previous results suggested that tyrosine kinases might serve as regulators of gamma-tubulin function, their exact roles remain enigmatic. In the present study, we show that a pool of gamma-tubulin associates with detergent-resistant membranes in differentiating P19 embryonal carcinoma cells, which exhibit elevated expression of the Src family kinase Fyn (protein tyrosine kinase p59(Fyn)). Microtubule-assembly assays demonstrated that membrane-associated gamma-tubulin complexes are capable of initiating the formation of microtubules. Pretreatment of the cells with Src family kinase inhibitors or wortmannin blocked the nucleation activity of the gamma-tubulin complexes. Immunoprecipitation experiments revealed that membrane-associated gamma-tubulin forms complexes with Fyn and PI3K (phosphoinositide 3-kinase). Furthermore, in vitro kinase assays showed that p85alpha (regulatory p85alpha subunit of PI3K) serves as a Fyn substrate. Direct interaction of gamma-tubulin with the C-terminal Src homology 2 domain of p85alpha was determined by pull-down experiments and immunoprecipitation experiments with cells expressing truncated forms of p85alpha. The combined results suggest that Fyn and PI3K might take part in the modulation of membrane-associated gamma-tubulin activities.     

Gamma-tubulin in Chlamydomonas: characterization of the gene and localization of the gene product in cells

In addition to their role in nucleating the assembly of axonemal microtubules, basal bodies often are associated with a microtubule organizing center (MTOC) for cytoplasmic microtubules. In an effort to define molecular components of the basal body apparatus in Chlamydomonas reinhardtii, genomic and cDNA clones encoding gamma-tubulin were isolated and sequenced. The gene, present in a single copy in the Chlamydomonas genome, encodes a protein with a predicted molecular mass of 52,161 D and 73% and 65% conservation with gamma-tubulin from higher plants and humans, respectively. To examine the distribution of gamma-tubulin in cells, a polyclonal antibody was raised against two peptides contained within the protein. Immunoblots of Chlamydomonas proteins show a major cross-reaction with a protein of Mr 53,000. In Chlamydomonas cells, the antibody stains the basal body apparatus as two or four spots at the base of the flagella and proximal to the microtubule rootlets. During cell division, two groups of fluorescent dots separate and localize to opposite ends of the mitotic apparatus. They then migrate during cleavage to positions known to be occupied by basal bodies. Changes in gamma-tubulin localization during the cell cycle are consistent with a role for this protein in the nucleation of microtubules of both the interphase cytoplasmic array and the mitotic spindle. Immunogold labeling of cell sections showed that gamma-tubulin is closely associated with the basal bodies. The flagellar transition region was also labeled, possibly indicating a role for gamma-tubulin in assembly of the central pair microtubules of the axoneme.     

Interactions between telomerase and primase physically link the telomere and chromosome replication machinery

In the ciliate Euplotes crassus, millions of new telomeres are synthesized by telomerase and polymerase alpha-primase during macronuclear development in mated cells. Concomitant with de novo telomere formation, telomerase assembles into higher-order complexes of 550 kDa, 1,600 kDa, and 5 MDa. We show here that telomerase is physically associated with the lagging-strand replication machinery in these complexes. Antibodies against DNA primase precipitated telomerase activity from all three complexes from mated cells but not the 280-kDa telomerase complex from vegetatively growing cells. Moreover, when telomerase was affinity purified, primase copurified with enzyme from mated cells but not with the 280-kDa vegetative complex. Thus, the association of telomerase and primase is developmentally regulated. Intriguingly, PCNA (proliferating cell nuclear antigen) was also found in the 5-MDa complex from mated cells. We therefore speculate that this complex is a complete telomere synthesis machine, while the smaller complexes are assembly intermediates. The physical association of telomerase and primase explains the coordinate regulation of telomeric G- and C-strand synthesis and the efficiency of telomere addition in E. crassus.     

Transient concentration of a gamma-tubulin-related protein with a pericentrin-related protein in the formation of basal bodies and flagella during the differentiation of Naegleria gruberi

The distribution of two proteins in Naegleria gruberi, N-gammaTRP (Naegleria gamma-tubulin-related protein) and N-PRP (Naegleria pericentrin-related protein), was examined during the de novo formation of basal bodies and flagella that occurs during the differentiation of N. gruberi. After the initiation of differentiation, N-gammaTRP and N-PRP began to concentrate at the same site within cells. The percentage of cells with a concentrated region of N-gammaTRP and N-PRP was maximal (68%) at 40 min when the synthesis of tubulin had just started but no assembled microtubules were visible. When concentrated tubulin became visible (60 min), the region of concentrated N-gammaTRP and N-PRP was co-localized with the tubulin spot and then flagella began to elongate from the region of concentrated tubulin. When cells had elongated flagella, the concentrated N-gammaTRP and N-PRP were translocated to the opposite end of the flagellated cells and disappeared. The transient concentration of N-gammaTRP coincided with the transient formation of an F-actin spot at which N-gammaTRP and alpha-tubulin mRNA were co-localized. The concentration of N-gammaTRP and formation of the F-actin spot occurred without the formation of microtubules but were inhibited by cytochalasin D. These observations suggest that the regional concentration of N-gammaTRP and N-PRP is mediated by actin filaments and might provide a site of microtubule nucleation for the assembly of newly synthesized tubulins into basal bodies and flagella.     

Arabidopsis GCP2 and GCP3 are part of a soluble gamma-tubulin complex and have nuclear envelope targeting domains

In higher plants, microtubules (MTs) are assembled in distinctive arrays in the absence of a defined organizing center. Three MT nucleation sites have been described: the nuclear surface, the cell cortex and cortical MT branch points. The Arabidopsis thaliana (At) genome contains putative orthologues encoding all the components of characterized mammalian nucleation complexes: gamma-tubulin and gamma-tubulin complex proteins GCP2 to GCP6. We have cloned the cDNA encoding AtGCP2, and show that gamma-tubulin, AtGCP2 and AtGCP3 are part of the same tandem affinity-purified complex and are present in a large membrane-associated complex. In addition, small soluble gamma-tubulin complexes of the size expected for a gamma-tubulin core complex are recruited to isolated nuclei. Using immunogold labelling, AtGCP3 is localized to both the nuclear envelope (NE) and the plasma membrane. To identify domains that could play a role in targeting complexes to these nucleation sites, truncated AtGCP2- and AtGCP3-green fluorescent protein fusion proteins were expressed in BY-2 cells. Several domains from AtGCP2 and AtGCP3 are capable of targeting fusions to the NE. We propose that regulated recruitment of soluble gamma-tubulin-containing complexes is responsible for nucleation at dispersed sites in plant cells and contributes to the formation and organization of the various MT arrays.     

De novo formation of centrosomes in vertebrate cells arrested during S phase

The centrosome usually replicates in a semiconservative fashion, i.e., new centrioles form in association with preexisting "maternal" centrioles. De novo formation of centrioles has been reported for a few highly specialized cell types but it has not been seen in vertebrate somatic cells. We find that when centrosomes are completely destroyed by laser microsurgery in CHO cells arrested in S phase by hydroxyurea, new centrosomes form by de novo assembly. Formation of new centrosomes occurs in two steps: approximately 5-8 h after ablation, clouds of pericentriolar material (PCM) containing gamma-tubulin and pericentrin appear in the cell. By 24 h, centrioles have formed inside of already well-developed PCM clouds. This de novo pathway leads to the formation of a random number of centrioles (2-14 per cell). Although clouds of PCM consistently form even when microtubules are completely disassembled by nocodazole, the centrioles are not assembled under these conditions.     

Plant gamma-tubulin interacts with alphabeta-tubulin dimers and forms membrane-associated complexes

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.     

Gamma-tubulin and MTOCs in Paramecium

The characterization of the two Paramecium gamma-tubulin genes, gammaPT1 and gammaPT2, allowed us to raise Paramecium-specific antibodies, directed against their most divergent carboxy-terminal peptide and to analyze the localization and dynamics of gamma-tubulin throughout the cell cycle. As in other cell types, a large proportion of the protein was found to be cytosolic, but in contrast to the general situation, gamma-tubulin was found to be permanently associated to four types of sites: basal bodies, the micronuclear compartment--within which mitotic and meiotic spindles develop without membrane breakdown, the pores of the contractile vacuoles and the cytoproct which are cortical microtubular organelles fulfilling excretory functions. In addition, a transient site of gamma-tubulin and microtubule assembly was observed at the site of nuclear exchange during conjugation. This complexity accounts for the nucleation of most of the numerous and diverse microtubule arrays present in Paramecium. The sites and mode of nucleation of the microtubule bundles formed in the macronuclear compartment during division remain unclear. These observations lead us to discuss the relationships between microtubules, gamma-tubulin and MTOCs.     

Basal body/centriole assembly and continuity

The long-standing interest in centrioles and basal bodies stems from the evolutionary conservation of their structural design and from their dual mode of assembly (templated versus de novo), revealed by electron microscopic studies nearly four decades ago and unique for a subcellular organelle. Molecular dissection of the assembly pathway during the past few years has recently progressed, essentially through direct and reverse genetic approaches. These studies revealed essential roles for centrins and the gamma-, delta-, epsilon - and eta-tubulins in assembly or as specific signals for centriole duplication. Identification of further components of basal bodies and centrioles might help to unravel the two assembly pathways and their regulation.     

Formation of basal bodies in the ciliary epithelium of molluscs Buccinum undatum L. and Lymnaea stagnalis L

Electron microscopic studies of the leg ciliary epithelium was carried out in two mollusks. In the epithelium of the leg of adult animals, the centrioles were mostly formed de novo with participation of deuterosomes during the formation of basal bodies. Transformation of the centriolar cylinder in a mature basal body is accompanied by the cylinder elongation and appearance of pericentriolar structures, such as rootlet system, basal legs, and basal plate. Centriolegenesis proceeds in both ciliate and nonciliate (with microvilli) cells of the epithelium. It has been proposed that the cell with microvilli represent a transitional stage in differentiation of the ciliary cells.