Gamma-tubulin distribution in the neuron: implications for the origins of neuritic microtubules

Axons and dendrites contain dense microtubule (MT) assays that are not attached to a traditional MT nucleating structure such as the centrosome. Nevertheless, the MTs within these neurites are highly organized with respect to their polarity, and consist of a regular 13-protofilament lattice, the two known characteristics of MTs nucleated at the centrosome. These observations suggest either that axonal and dendritic MTs arise at the centrosome, or that they are nucleated locally, following a redistribution of MT nucleating material from the centrosome during neuronal development. To begin distinguishing between these possibilities, we have determined the distribution of gamma-tubulin within cultured sympathetic neurons. gamma-tubulin, a newly discovered protein which is specifically localized to the pericentriolar region of nonneuronal cells (Zheng, Y., M. K. Jung, and B. R. Oakley. 1991. Cell. 65:817-823; Stearns, T., L. Evans, and M. Kirschner. 1991. Cell. 65:825-836), has been shown to play a critical role in MT nucleation in vivo (Joshi, H. C., M. J. Palacios, L. McNamara, and D. W. Cleveland. 1992. Nature (Lond.). 356:80-83). Because the gamma-tubulin content of individual cells is extremely low, we relied principally on the high degree of resolution and sensitivity afforded by immunoelectron microscopy. Our studies reveal that, like the situation in nonneuronal cells, gamma-tubulin is restricted to the pericentriolar region of the neuron. Furthermore, serial reconstruction analyses indicate that the minus ends of MTs in both axons and dendrites are free of gamma-tubulin immunoreactivity. The absence of gamma-tubulin from the axon was confirmed by immunoblot analyses of pure axonal fractions obtained from explant cultures. The observation that gamma-tubulin is restricted to the pericentriolar region of the neuron provides compelling support for the notion that MTs destined for axons and dendrites are nucleated at the centrosome, and subsequently released for translocation into these neurites.     

CP60: a microtubule-associated protein that is localized to the centrosome in a cell cycle-specific manner.

DMAP190 is a microtubule-associated protein from Drosophila that is localized to the centrosome. In a previous study, we used affinity chromatography to identify proteins that interact with DMAP190, and identified a 60-kDa protein that we named DMAP60 (Kellogg and Alberts, 1992). Like DMAP190, DMAP60 interacts with microtubules and is localized to the centrosome, and the two proteins associate as part of a multiprotein complex. We now report the cloning and sequencing of the cDNA encoding DMAP60. The amino acid sequence of DMAP60 is not homologous to any protein in the database, although it contains six consensus sites for phosphorylation by cyclin-dependent kinases. As judged by in situ hybridization, the gene for DMAP60 maps to chromosomal region 46A. In agreement with others working on Drosophila centrosomal proteins, we have changed the names for DMAP190 and DMAP60 to CP190 and CP60, respectively, to give these proteins a consistent nomenclature. Antibodies that recognize CP60 reveal that it is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is maximal during anaphase and telophase, and then drops dramatically during late telophase or early interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase. CP60 isolated from Drosophila embryos is highly phosphorylated, and dephosphorylated CP60 is a good substrate for cyclin B/p34cdc2 kinase complexes. A second kinase activity capable of phosphorylating CP60 is present in the CP60/CP190 multiprotein complex. We find that bacterially expressed CP60 binds to purified microtubules, and this binding is blocked by CP60 phosphorylation.     

The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19- amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.     

The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is gamma-tubulin dependent

gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.     

GCP-WD is a gamma-tubulin targeting factor required for centrosomal and chromatin-mediated microtubule nucleation

The gamma-tubulin ring complex (gammaTuRC) is a large multi-protein complex that is required for microtubule nucleation from the centrosome. Here, we show that the GCP-WD protein (originally named NEDD1) is the orthologue of the Drosophila Dgrip71WD protein, and is a subunit of the human gammaTuRC. GCP-WD has the properties of an attachment factor for the gammaTuRC: depletion or inhibition of GCP-WD results in loss of the gammaTuRC from the centrosome, abolishing centrosomal microtubule nucleation, although the gammaTuRC is intact and able to bind to microtubules. GCP-WD depletion also blocks mitotic chromatin-mediated microtubule nucleation, resulting in failure of spindle assembly. Mitotic phosphorylation of GCP-WD is required for association of gamma-tubulin with the spindle, separately from association with the centrosome. Our results indicate that GCP-WD broadly mediates targeting of the gammaTuRC to sites of microtubule nucleation and to the mitotic spindle, which is essential for spindle formation.     

Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules

gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.     

Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.     

Reorganization of microtubule nucleation during muscle differentiation

Skeletal muscle differentiation involves a complete reorganization of the microtubule network. Nearly 20 years ago, Tassin et al. [1985: J Cell Biol 100:35-46] suggested a mechanism for this reorganization by showing a redistribution of the microtubule organizing center from the centrosome to the nuclear membrane. Little progress has been made since. It is still not clear whether centrosomal proteins are redistributed together, whether microtubules are nucleated at the nuclear membrane or transported there post-nucleation, and whether gamma-tubulin (gammatub) remains necessary for nucleation in myotubes. To investigate these questions, we have examined the redistribution of the centrosomal proteins pericentrin (PC), gammatub, and ninein in the C2 muscle cell line. Immunofluorescence of differentiated myotubes shows PC along the nuclear membrane whereas gammatub is only detected there after pre-fixation detergent extraction. After expression of a GFP-tagged gammatub, we observe a weak fluorescence along the nuclear membrane, confirming the presence of gammatub at a low concentration relative to PC. Microinjection of anti-gammatub antibodies into myotubes blocks microtubule growth from both nuclear membranes and centrosomal sites. The centrosomal microtubule-anchoring protein, ninein, is found at the nuclear membrane as well and its distribution appears independent of microtubule integrity. We conclude that centrosomal proteins are redistributed independently during muscle differentiation, to sites that nucleate microtubules both along the nuclear membranes and through the cytoplasm.     

Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies.

A 190-kDa centrosomal protein interacts with microtubules when Drosophila embryo extracts are passed over microtubule-affinity columns. We have obtained a partial cDNA clone that encodes this protein. Using a fusion protein produced from the clone, we have developed a novel immunoaffinity chromatography procedure that allows both the 190-kDa protein and a complex of proteins that associates with it to be isolated in in a single step. For this procedure, the fusion protein is used as an antigen to prepare rabbit polyclonal antibodies, and those antibodies that recognize the 190-kDa protein with low affinity are selectively purified on a column containing immobilized antigen. These low-affinity antibodies are then used to construct an immunoaffinity column. When Drosophila embryo extracts are passed over this column, the 190-kDa protein is quantitatively retained and can be eluted in nearly pure form under nondenaturing conditions with 1.5 M MgCl2, pH 7.6. The immunoaffinity column is washed with 1.0 M KCl just before the elution with 1.5 M MgCl2. This wash elutes 10 major proteins, as well as a number of minor ones. We present evidence that these KCl-eluted proteins represent additional centrosomal components that interact with the 190-kDa protein to form a multiprotein complex within the cell.     

Desmoplakin: an unexpected regulator of microtubule organization in the epidermis

Despite their importance in cell shape and polarity generation, the organization of microtubules in differentiated cells and tissues remains relatively unexplored in mammals. We generated transgenic mice in which the epidermis expresses a fluorescently labeled microtubule-binding protein and show that in epidermis and in cultured keratinocytes, microtubules stereotypically reorganize as they differentiate. In basal cells, microtubules form a cytoplasmic network emanating from an apical centrosome. In suprabasal cells, microtubules concentrate at cell-cell junctions. The centrosome retains its ability to nucleate microtubules in differentiated cells, but no longer anchors them. During epidermal differentiation, ninein, which is a centrosomal protein required for microtubule anchoring (Dammermann, A., and A. Merdes. 2002. J. Cell Biol. 159:255-266; Delgehyr, N., J. Sillibourne, and M. Bornens. 2005. J. Cell Sci. 118:1565-1575; Mogensen, M.M., A. Malik, M. Piel, V. Bouckson-Castaing, and M. Bornens. 2000. J. Cell Sci. 113:3013-3023), is lost from the centrosome and is recruited to desmosomes by desmoplakin (DP). Loss of DP prevents accumulation of cortical microtubules in vivo and in vitro. Our work uncovers a differentiation-specific rearrangement of the microtubule cytoskeleton in epidermis, and defines an essential role for DP in the process.     

Dynamics of microtubules and positioning of female pronucleus during bovine parthenogenesis

The zygote centrosome, consisting of both paternal and maternal centrosomal components, is the microtubule-organizing center necessary for pronuclear migration and positioning in fertilization. Maternal centrosomal function in microtubule organization and pronuclear positioning, however, remains unclear. In the present study, we sought to elucidate the function of maternal centrosomes during bovine parthenotes in the microtubule organizational processes required to move the pronucleus to the cell center without sperm centrosomal components. Microtubule organization, pronuclear position, and distribution of gamma-tubulin, which is thought to be the major component of maternal centrosomal material, were imaged by immunocytochemistry and conventional epifluorescence microscopy. In bovine parthenotes treated with paclitaxel, a microtubule-stabilizing drug, the cytoplasmic microtubule asters became organized after chemical activation, and the microtubules radiated dynamically toward the female pronucleus. The microtubule patterns correlated well with pronuclear movement to the cell center. Microtubules aggregated at regions of gamma-tubulin concentration, but gamma-tubulin did not localize to a spot until the first interphase of bovine parthenogenesis. These findings indicate that gamma-tubulin is responsible for microtubule organization as the maternal centrosome. In bovine parthenogenesis, the maternal centrosome then organizes cytoplasmic microtubules to move the female pronucleus into the cell center. We propose that the maternal centrosome plays a role as a functional centrosome despite the lack of a sperm contribution, making this structure less competent for microtubule organization in comparison with centrosomes containing sperm centrosomal components.     

Microtubule polarities indicate that nucleation and capture of microtubules occurs at cell surfaces in Drosophila

Hook decoration with pig brain tubulin was used to assess the polarity of microtubules which mainly have 15 protofilaments in the transcellular bundles of late pupal Drosophila wing epidermal cells. The microtubules make end-on contact with cell surfaces. Most microtubules in each bundle exhibited a uniform polarity. They were oriented with their minus ends associated with their hemidesmosomal anchorage points at the apical cuticle-secreting surfaces of the cells. Plus ends were directed towards, and were sometimes connected to, basal attachment desmosomes at the opposite ends of the cells. The orientation of microtubules at cell apices, with minus ends directed towards the cell surface, is opposite to the polarity anticipated for microtubules which have elongated centrifugally from centrosomes. It is consistent, however, with evidence that microtubule assembly is nucleated by plasma membrane-associated sites at the apical surfaces of the cells (Mogensen, M. M., and J. B. Tucker. 1987. J. Cell Sci. 88:95-107) after these cells have lost their centriole-containing, centrosomal, microtubule-organizing centers (Tucker, J. B., M. J. Milner, D. A. Currie, J. W. Muir, D. A. Forrest, and M.-J. Spencer. 1986. Eur. J. Cell Biol. 41:279-289). Our findings indicate that the plus ends of many of these apically nucleated microtubules are captured by the basal desmosomes. Hence, the situation may be analogous to the polar-nucleation/chromosomal-capture scheme for kinetochore microtubule assembly in mitotic and meiotic spindles. The cell surface-associated nucleation-elongation-capture mechanism proposed here may also apply during assembly of transcellular microtubule arrays in certain other animal tissue cell types.     

Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle

Since the discovery of gamma-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of gamma-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for gamma-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2-6) required for localizing gamma-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for gamma-tubulin may lie within the spindle, where augmin and gamma-tubulin function cooperatively to amplify the number of microtubules.     

Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma- tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.     

NEDD1-dependent recruitment of the gamma-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly

The centrosome is the major microtubule organizing structure in somatic cells. Centrosomal microtubule nucleation depends on the protein gamma-tubulin. In mammals, gamma-tubulin associates with additional proteins into a large complex, the gamma-tubulin ring complex (gammaTuRC). We characterize NEDD1, a centrosomal protein that associates with gammaTuRCs. We show that the majority of gammaTuRCs assemble even after NEDD1 depletion but require NEDD1 for centrosomal targeting. In contrast, NEDD1 can target to the centrosome in the absence of gamma-tubulin. NEDD1-depleted cells show defects in centrosomal microtubule nucleation and form aberrant mitotic spindles with poorly separated poles. Similar spindle defects are obtained by overexpression of a fusion protein of GFP tagged to the carboxy-terminal half of NEDD1, which mediates binding to gammaTuRCs. Further, we show that depletion of NEDD1 inhibits centriole duplication, as does depletion of gamma-tubulin. Our data suggest that centriole duplication requires NEDD1-dependent recruitment of gamma-tubulin to the centrosome.     

Structure of the gamma-tubulin ring complex: a template for microtubule nucleation

The gamma-tubulin ring complex (gammaTuRC) is a protein complex of relative molecular mass approximately 2.2 x 10(6) that nucleates microtubules at the centrosome. Here we use electron-microscopic tomography and metal shadowing to examine the structure of isolated Drosophila gammaTuRCs and the ends of microtubules nucleated by gammaTuRCs and by centrosomes. We show that the gammaTuRC is a lockwasher-like structure made up of repeating subunits, topped asymmetrically with a cap. A similar capped ring is also visible at one end of microtubules grown from isolated gammaTuRCs and from centrosomes. Antibodies against gamma-tubulin label microtubule ends, but not walls, in centrosomes. These data are consistent with a template-mediated mechanism for microtubule nucleation by the gammaTuRC.     

Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.     

Interaction of an Overexpressed gamma-Tubulin with Microtubules In Vivo and In Vitro

gamma-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for gamma-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus gamma-tubulin. When CHO cells were transiently transfected with the gamma-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such gamma-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that gamma-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas gamma-tubulin purified from insect Sf9 cells (), interaction between gamma-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with gamma-tubulin monomers in vitro were associated with more gold particles conjugated with gamma-tubulin than in controls where no exogenous gamma-tubulin was added. However, binding of gamma-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although gamma-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (; ), which might be ascribed to the difference in the level of protein expression in transfected cells.     

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.