Microtubule nucleation at non-spindle pole body microtubule-organizing centers requires fission yeast centrosomin-related protein mod20p

BACKGROUND: Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized. In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent. These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase. RESULTS: From an insertional mutagenesis screen we identified a novel gene, mod20+, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast. Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis. Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves. We find that mod20p is required for the localization of components of the gamma-tubulin complex to non-spindle pole body MTOCs and physically interacts with the gamma-tubulin complex in vivo. Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin. CONCLUSIONS: Mod20p appears to act by recruiting components of the gamma-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells.     

GCP6 binds to intermediate filaments: a novel function of keratins in the organization of microtubules in epithelial cells

In simple epithelial cells, attachment of microtubule-organizing centers (MTOCs) to intermediate filaments (IFs) enables their localization to the apical domain. It is released by cyclin-dependent kinase (Cdk)1 phosphorylation. Here, we identified a component of the gamma-tubulin ring complex, gamma-tubulin complex protein (GCP)6, as a keratin partner in yeast two-hybrid assays. This was validated by binding in vitro of both purified full-length HIS-tagged GCP6 and a GCP6(1397-1819) fragment to keratins, and pull-down with native IFs. Keratin binding was blocked by Cdk1-mediated phosphorylation of GCP6. GCP6 was apical in normal enterocytes but diffuse in K8-null cells. GCP6 knockdown with short hairpin RNAs (shRNAs) in CACO-2 cells resulted in gamma-tubulin signal scattered throughout the cytoplasm, microtubules (MTs) in the perinuclear and basal regions, and microtubule-nucleating activity localized deep in the cytoplasm. Expression of a small fragment GCP6(1397-1513) that competes binding to keratins in vitro displaced gamma-tubulin from the cytoskeleton and resulted in depolarization of gamma-tubulin and changes in the distribution of microtubules and microtubule nucleation sites. Expression of a full-length S1397D mutant in the Cdk1 phosphorylation site delocalized centrosomes. We conclude that GCP6 participates in the attachment of MTOCs to IFs in epithelial cells and is among the factors that determine the peculiar architecture of microtubules in polarized epithelia.     

Fission yeast mto2p regulates microtubule nucleation by the centrosomin-related protein mto1p

From an insertional mutagenesis screen, we isolated a novel gene, mto2+, involved in microtubule organization in fission yeast. mto2Delta strains are viable but exhibit defects in interphase microtubule nucleation and in formation of the postanaphase microtubule array at the end of mitosis. The mto2Delta defects represent a subset of the defects displayed by cells deleted for mto1+ (also known as mod20+ and mbo1+), a centrosomin-related protein required to recruit the gamma-tubulin complex to cytoplasmic microtubule-organizing centers (MTOCs). We show that mto2p colocalizes with mto1p at MTOCs throughout the cell cycle and that mto1p and mto2p coimmunoprecipitate from cytoplasmic extracts. In vitro studies suggest that mto2p binds directly to mto1p. In mto2Delta mutants, although some aspects of mto1p localization are perturbed, mto1p can still localize to spindle pole bodies and the cell division site and to "satellite" particles on interphase microtubules. In mto1Delta mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. We also find that in mto2Delta mutants, cytoplasmic forms of the gamma-tubulin complex are mislocalized, and the gamma-tubulin complex no longer coimmunoprecipitates with mto1p from cell extracts. These experiments establish mto2p as a major regulator of mto1p-mediated microtubule nucleation by the gamma-tubulin complex.     

Differential properties of the two Drosophila gamma-tubulin isotypes.

The functional significance of distinct gamma-tubulins in several unrelated eukaryotes remains an enigma due to the difficulties to investigate this question experimentally. Using specific nucleotidic and immunological probes, we have demonstrated that the two divergent Drosophila gamma-tubulins, gamma-tub23C and gamma-tub37CD, are expressed in cultured cells. Gamma-tub37CD is constantly detected at the centrosome and absent in the mitotic spindle, while gamma-tub23C is extensively recruited to the centrosome during mitosis and relocalizes in the mitotic spindle. The two gamma-tubulins exhibit distinct biochemical properties. Gamma-tub23C is present in the soluble gamma-tubulin small complexes (10S) and gamma-tubulin big complexes (35S) and is loosely associated to the cytoskeleton. In contrast, gamma-tub37CD is undetectable in the soluble fraction and exhibits a tight binding to the centrosome. Syncytial embryos also contain the two gamma-tubulin isotypes, which are differentially recruited at the centrosome. Gamma-tub23C is present in the 10S soluble complexes only, while y-tub37CD is contained in the two soluble complexes and is recruited at the centrosome where it exhibits an heterogeneous binding. These results demonstrated an heterogeneity of the two Drosophila gamma-tubulin isotypes both in the cytoskeletal and the soluble fractions. They suggest the direct implication of the 35S complex in the centrosomal recruitment of gamma-tubulin and a conditional functional redundancy between the two gamma-tubulins.     

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 is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans

We have recently discovered that the mipA gene of A. nidulans encodes gamma-tubulin, a new member of the tubulin superfamily. To determine the function of gamma-tubulin in vivo, we have created a mutation in the mipA gene by integrative transformation, maintained the mutation in a heterokaryon, and determined the phenotype of the mutation in spores produced by the heterokaryon. The mutation is lethal and recessive. It strongly inhibits nuclear division, less strongly inhibits nuclear migration, and, as judged by immunofluorescence microscopy, causes a reduction in the number and length of cytoplasmic microtubules and virtually a complete absence of mitotic apparatus. We conclude that gamma-tubulin is essential for microtubule function in general and nuclear division in particular. Immunofluorescence microscopy of wild-type hyphae with affinity-purified, gamma-tubulin-specific antibodies reveals that gamma-tubulin is a component of interphase and mitotic spindle pole bodies. We propose that gamma-tubulin attaches microtubules to the spindle pole body, nucleates microtubule assembly, and establishes microtubule polarity in vivo.     

Mto2p, a novel fission yeast protein required for cytoplasmic microtubule organization and anchoring of the cytokinetic actin ring

Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires gamma-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Delta cells fail to establish a stable EMTOC and localize gamma-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Delta cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Delta cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis.     

Gamma-tubulin in basal land plants: characterization, localization, and implication in the evolution of acentriolar microtubule organizing centers

Although seed plants have gamma-tubulin, a ubiquitous component of centrosomes associated with microtubule nucleation in algal and animal cells, they do not have discrete microtubule organizing centers (MTOCs) comparable to animal centrosomes, and the organization of microtubule arrays in plants has remained enigmatic. Spindle development in basal land plants has revealed a surprising variety of MTOCs that may represent milestones in the evolution of the typical diffuse acentrosomal plant spindle. We have isolated and characterized the gamma-tubulin gene from a liverwort, one of the extant basal land plants. Sequence similarity to the gamma-tubulin gene of higher plants suggests that the gamma-tubulin gene is highly conserved in land plants. The G9 antibody to fission yeast gamma-tubulin recognized a single band of 55 kD in immunoblots from bryophytes. Immunohistochemistry with the G9 antibody clearly documented the association of gamma-tubulin with various MTOC sites in basal land plants (e.g., discrete centrosomes with and without centrioles and the plastid surface in monoplastidic meiosis of bryophytes). Changes in the distribution of gamma-tubulin occur in a cell cycle-specific manner during monoplastidic meiosis in the liverwort Dumortiera hirsuta. gamma-Tubulin changes its localization from the plastid surface in prophase I to the spindle, from the spindle to phragmoplasts and the nuclear envelope in telophase I, and back to the plastid surfaces in prophase II. In vitro experiments show that gamma-tubulin is detectable on the surface of isolated plastids and nuclei of D. hirsuta, and microtubules can be repolymerized from the isolated plastids. gamma-Tubulin localization patterns on plastid and nuclear surfaces are not affected by the destruction of microtubules by oryzalin. We conclude that gamma-tubulin is a highly conserved protein associated with microtubule nucleation in basal land plants and that it has a cell cycle-dependent distribution essential for the orderly succession of microtubule arrays.     

Characterization of microsome-associated tobacco BY-2 centrins

Centrin - higher plants - MTOCs - microtubules nucleation In most eukaryotic cells, the Ca(2+)-binding protein centrin is associated with structured microtubule-organizing centers (MTOCs) such as centrosomes. In these cells, centrin either forms centrosome-associated contractile fibers, or is involved in centrosome biogenesis. Our aim was to investigate the functions of centrin in higher plant cells which do not contain centrosome-like MTOCs. We have cloned two tobacco BY-2 centrin cDNAs and we show that higher plant centrins define a phylogenetic group of proteins distinct from centrosome-associated centrins. In addition, tobacco centrins were found primarily associated with microsomes and did not colocalize with gamma-tubulin, a known MTOC marker. While the overall level of centrin did not vary during the cell cycle, centrin was prominently detected at the cell plate during telophase. Our results suggest that in tobacco, the major portion of centrin is not MTOC-associated and could be involved in the formation of the cell plate during cytokinesis.     

Induction of centrosome injury, multipolar spindles and multipolar division in cultured V79 cells exposed to dimethylarsinic acid: role for microtubules in centrosome dynamics

Role for microtubules in the induction of multiple microtubule organizing centers (MTOCs) and multipolar spindles by dimethylarsinic acid (DMAA), a methylated derivative of inorganic arsenics, was investigated with respect to the effects of microtubule disruption and reorganization. DMAA induced multiple signals of gamma-tubulin, a well-characterized component of MTOCs in the centrosome, in a manner specific to mitotic cells. The multiple signals of gamma-tubulin were co-localized with multipolar spindles caused by DMAA. Disruption of microtubules by nocodazole (NOZ) suppressed the appearance of centrosome injury caused by DMAA while disorganization of actin microfilaments by cytochalasin D did not. Post-treatment incubation of cells in which multiple signals of gamma-tubulin caused by DMAA had been coalesced to one or two dots by NOZ caused the reappearance of mitotic cells with multiple signals of gamma-tubulin, in conjunction with reorganization of the microtubules. These results suggest a role for microtubules in the dynamic behavior of the mitotic centrosome. DMAA induced aberrant cytokinesis, such as tripolar and quadripolar division, in a concentration-dependent manner. These results, together with the findings of earlier studies, suggest that the centrosome is the primary target for the induction of multipolar spindles by DMAA and the resultant induction of multinucleation and multipolar division.     

Receptors determine the cellular localization of a gamma-tubulin complex and thereby the site of microtubule formation.

The yeast microtubule organizing centre (MTOC), known as the spindle pole body (SPB), organizes the nuclear and cytoplasmic microtubules which are functionally and spatially distinct. Microtubule organization requires the yeast gamma-tubulin complex (Tub4p complex) which binds to the nuclear side of the SPB at the N-terminal domain of Spc110p. Here, we describe the identification of the essential SPB component Spc72p whose N-terminal domain interacts with the Tub4p complex on the cytoplasmic side of the SPB. We further report that this Tub4p complex-binding domain of Spc72p is essential and that temperature-sensitive alleles of SPC72 or overexpression of a binding domain-deleted variant of SPC72 (DeltaN-SPC72) impair cytoplasmic microtubule formation. Consequently, polynucleated and anucleated cells accumulated in these cultures. In contrast, overexpression of the entire SPC72 results in more cytoplasmic microtubules compared with wild-type. Finally, exchange of the Tub4p complex-binding domains of Spc110p and Spc72p established that the Spc110p domain, when attached to DeltaN-Spc72p, was functional at the cytoplasmic site of the SPB, while the corresponding domain of Spc72p fused to DeltaN-Spc110p led to a dominant-negative effect. These results suggest that different components of MTOCs act as receptors for gamma-tubulin complexes and that they are essential for the function of MTOCs.     

Gamma-tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells

Cortical microtubules are considered to regulate the direction of cellulose microfibril deposition. Despite their significant role in determining cell morphology, cortical microtubules completely disappear from the cell cortex during M phase and become reorganized at G1 phase. The mechanism by which these microtubules become properly formed again is, however, still unclear. We have proposed that the origin of cortical microtubules is on the daughter nuclear surface, but further cortical microtubule reorganization occurs at the cell cortex. Hence it is probable that the locations of microtubule organizing centers (MTOCs) are actively changing. However, the actual MTOC sites of cortical microtubules were not clearly determined. In this paper, we have examined the distribution of gamma-tubulin, one of the key molecules of MTOCs in various organisms, during cortical microtubule reorganization using both immunofluorescence and a GFP reporter system. Using a monoclonal antibody (clone G9) that recognizes highly conserved residues in y-tubulin, y-tubulin was found to be constitutively expressed and to be clearly localized to microtubule structures, such as the preprophase bands, spindles, and phragmoplasts, specific to each cell cycle stage. This distribution pattern was confirmed by the GFP reporter system. During cortical microtubule reorganization at the M to G1 transition phase, gamma-tubulin first accumulated at the daughter nuclear surfaces, and then seemed to spread onto the cell cortex along with microtubules elongating from the daughter nuclei. Based on the results, it was confirmed that daughter nuclear surfaces acted as origins of cortical microtubules, and that further reorganization occurred on the cell cortex.     

Spindle Dynamics and the Role of γ-Tubulin in Early Caenorhabditis elegans Embryos

gamma-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that gamma-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans gamma-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::gamma-tubulin or GFP::beta-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of gamma-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of gamma-tubulin. gamma-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that gamma-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.     

Gamma-tubulin and the C-terminal motor domain kinesin-like protein, KLPA, function in the establishment of spindle bipolarity in Aspergillus nidulans

Previous research has found that a gamma-tubulin mutation in Schizosaccharomyces pombe is synthetically lethal with a deletion of the C-terminal motor domain kinesin-like protein gene pkl1, but the lethality of the double mutant prevents a phenotypic analysis of the synthetic interaction. We have investigated interactions between klpA1, a deletion of an Aspergillus nidulans homolog of pkl1, and mutations in the mipA, gamma-tubulin gene. We find that klpA1 dramatically increases the cold sensitivity and slightly reduces the growth rate at all temperatures, of three mipA alleles. In synchronized cells we find that klpA1 causes a substantial but transient inhibition of the establishment of spindle bipolarity. At a restrictive temperature, mipAD123 causes a slight, transient inhibition of spindle bipolarity and a more significant inhibition of anaphase A. In the mipAD123/klpA1 strain, formation of bipolar spindles is more strongly inhibited than in the klpA1 single mutant and many spindles apparently never become bipolar. These results indicate, surprisingly, that gamma-tubulin and the klpA kinesin have overlapping roles in the establishment of spindle bipolarity. We propose a model to account for these data.     

BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin

BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.     

Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes

To better understand the differences in cytoskeletal organization between in vivo (IVO) and in vitro (IVM) matured oocytes, we analyzed remodeling of the centrosome-microtubule complex in IVO and IVM mouse oocytes. Fluorescence imaging revealed dramatic differences in meiotic spindle assembly and organization between these two populations. Metaphase spindles at both meiosis I (M-I) and meiosis II (M-II) in IVO oocytes were compact, displayed focused spindle poles with distinct gamma-tubulin foci, and were composed of acetylated microtubules. In contrast, IVM oocytes exhibited barrel-shaped spindles with fewer acetylated microtubules and gamma-tubulin diffusely distributed throughout the spindle proper. With respect to meiotic progression, IVO oocytes were more synchronous in the rate and extent of anaphase to telophase of M-I and first polar body emission than were IVM counterparts. Furthermore, IVO oocytes showed a twofold increase in cytoplasmic microtubule organizing centers (MTOCs), and constitutive MTOC proteins (gamma-tubulin and pericentrin) were excluded from the first polar body. Inclusion of MTOC constitutive proteins in the polar body and diminished number of cytoplasmic MTOCs was observed in IVM oocytes. These findings were corroborated in IVO oocytes obtained from naturally ovulated and spontaneously cycling mice and highlight a fundamental distinction in the spatial and temporal regulation of microtubule dynamics between IVO and IVM oocytes     

Rsp1p, a J domain protein required for disassembly and assembly of microtubule organizing centers during the fission yeast cell cycle

Regulation of microtubule organizing centers (MTOCs) orchestrates the reorganization of the microtubule (MT) cytoskeleton. In the fission yeast Schizosaccharomyces pombe, an equatorial MTOC (eMTOC) at the cell division site disassembles after cytokinesis, and multiple interphase MTOCs (iMTOCs) appear on the nucleus. Here, we show that, upon eMTOC disassembly, small satellites carrying MTOC components such as the gamma-tubulin complex travel in both directions along interphase MTs. We identify rsp1p, an MTOC protein required for eMTOC disassembly. In rsp1 loss-of-function mutants, the eMTOC persists and organizes an abnormal microtubule aster, while iMTOCs and satellites are greatly reduced. Conversely, rsp1p overexpression inhibits eMTOC formation. Rsp1p is a J domain protein that interacts with an hsp70. Thus, our findings suggest a model in which rsp1p is part of a chaperone-based mechanism that disassembles the eMTOC into satellites, contributing to the dynamic redistribution of MTOC components for organization of interphase microtubules.     

The abnormal spindle protein is required for germ cell mitosis and oocyte differentiation during Drosophila oogenesis

In this study, we present evidence that the asp function is required in oogenesis for germline cell divisions as well as for cyst polarity and oocyte differentiation. Consistent with previously described roles in spindle organization during Drosophila meiosis and mitosis, asp mutation leads to severe defects in spindle microtubule organization within the germarium. The mitotic spindles of the mutant cystocytes are composed by wavy microtubules and have abnormal poles that often lack gamma-tubulin. The fusome structure is also compromised. In the absence of asp function, the cystocyte divisions fail resulting in egg chamber with fewer than 16 germ cells. Moreover, the microtubule network within the developing germline cysts may assemble incorrectly in turn affecting the microtubule based transport of the specific determinants that is required during mid-oogenesis for the oocyte differentiation program.     

Polo-like kinase 1 regulates Nlp,a centrosome protein involved in microtubule nucleation

In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.     

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.