Centrosome duplication. a centriolar pas de deux.
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Animals↗
Biomedical subjects
T Stearns
Publications and source records attributed to T Stearns.
GCP5 and GCP6: two new members of the human gamma-tubulin complex.
The gamma-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human gamma-tubulin complex and the identification of its subunits. The human gamma-tubulin complex is a ring of ~25 nm, has a subunit structure similar to that reported for gamma-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human gamma-tubulin complex components confirmed the presence of four previously identified components (gamma-tubulin and gamma-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the gamma-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire gamma-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of gamma-tubulin, GCP2, GCP3, and GCP4 within the gamma-tubulin complex. Thus, the gamma-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.
Amino Acid Sequence↗
The DNA damage checkpoint signal in budding yeast is nuclear limited.
The nature of the DNA damage-induced checkpoint signal that causes the arrest of cells prior to mitosis is unknown. To determine if this signal is transmitted through the cytoplasm or is confined to the nucleus, we created binucleate heterokaryon yeast cells in which one nucleus suffered an unrepairable double-strand break, and the second nucleus was undamaged. In most of these binucleate cells, the damaged nucleus arrested prior to spindle elongation, while the undamaged nucleus completed mitosis, even when the strength of the damage signal was increased. The arrest of the damaged nucleus was dependent upon the function of the RAD9 checkpoint gene. Thus, the DNA damage checkpoint causing G2/M arrest is regulated by a signal that is nuclear limited.
Cell Cycle↗
Delta-tubulin and epsilon-tubulin: two new human centrosomal tubulins reveal new aspects of centrosome structure and function.
The centrosome organizes microtubules, which are made up of alpha-tubulin and beta-tubulin, and contains centrosome-bound gamma-tubulin, which is involved in microtubule nucleation. Here we identify two new human tubulins and show that they are associated with the centrosome. One is a homologue of the Chlamydomonas delta-tubulin Uni3, and the other is a new tubulin, which we have named epsilon-tubulin. Localization of delta-tubulin and epsilon-tubulin to the centrosome is independent of microtubules, and the patterns of localization are distinct from each other and from that of gamma-tubulin. Delta-tubulin is found in association with the centrioles, whereas epsilon-tubulin localizes to the pericentriolar material. epsilon-Tubulin exhibits a cell-cycle-specific pattern of localization, first associating with only the older of the centrosomes in a newly duplicated pair and later associating with both centrosomes. epsilon-Tubulin thus distinguishes the old centrosome from the new at the level of the pericentriolar material, indicating that there may be a centrosomal maturation event that is marked by the recruitment of epsilon-tubulin.
Antineoplastic Agents↗
Arrest, adaptation, and recovery following a chromosome double-strand break in Saccharomyces cerevisiae.
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Cell Cycle↗
Components of an SCF ubiquitin ligase localize to the centrosome and regulate the centrosome duplication cycle.
Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, we show that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. We identify candidate centrosomal F-box proteins, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle.
3T3 Cells↗
Cyclin-dependent kinase control of centrosome duplication.
Centrosomes nucleate microtubules and duplicate once per cell cycle. This duplication and subsequent segregation in mitosis results in maintenance of the one centrosome/cell ratio. Centrosome duplication occurs during the G1/S transition in somatic cells and must be coupled to the events of the nuclear cell cycle; failure to coordinate duplication and mitosis results in abnormal numbers of centrosomes and aberrant mitoses. Using both in vivo and in vitro assays, we show that centrosome duplication in Xenopus laevis embryos requires cyclin/cdk2 kinase activity. Injection of the cdk (cyclin-dependent kinase) inhibitor p21 into one blastomere of a dividing embryo blocks centrosome duplication in that blastomere; the related cdk inhibitor p27 has a similar effect. An in vitro system using Xenopus extracts carries out separation of the paired centrioles within the centrosome. This centriole separation activity is dependent on cyclin/cdk2 activity; depletion of either cdk2 or of the two activating cyclins, cyclin A and cyclin E, eliminates centriole separation activity. In addition, centriole separation is inhibited by the mitotic state, suggesting a mechanism of linking the cell cycle to periodic duplication of the centrosome.
Animals↗
Alf1p, a CLIP-170 domain-containing protein, is functionally and physically associated with alpha-tubulin.
Tubulin is a heterodimer of alpha- and beta-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y. Huang, H. Rommelaere, J. Vandekerckhove, C. Ampe, and N.J. Cowan. 1996. Cell. 86:287-296; Tian, G., S.A. Lewis, B. Feierbach, T. Stearns, H. Rommelaere, C. Ampe, and N.J. Cowan. 1997. J. Cell Biol. 138:821-832). We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B. Alf1p interacts with alpha-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with alpha-tubulin. Mutations in alpha-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of alpha-tubulin; this domain is distinct from the region of interaction between alpha-tubulin and beta-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in alpha-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast alpha-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional alpha-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester alpha-tubulin from interaction with beta-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.
Amino Acid Sequence↗
Cytoskeletal dynamics in yeast.
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Animals↗
Gamma-tubulin complexes: size does matter.
gamma-Tubulin is a conserved component of all microtubule-organizing centres and is required for these organelles to nucleate microtubule polymerization. However, the mechanism of nucleation is not known. In addition to its localization to organizing centres, a large pool of gamma-tubulin exists in the cytoplasm in a complex with other proteins. The size of the gamma-tubulin complex and number of associated proteins vary among organisms, and the functional significance of these differences is unknown. Recently, the nature of these gamma-tubulin complexes has been explored in different organisms, and this has led us closer to a molecular understanding of microtubule nucleation.
Animal Population Groups↗
Centrosome reduction during mouse spermiogenesis.
The sperm does not contribute the centrosome during murine fertilization. To determine the manner in which a functional centrosome is reduced, we have studied centrosome degeneration during spermiogenesis of mice. The round spermatids display normal centrosomes consisting of a pair of centrioles along with gamma-tubulin containing foci. However, they do not seem to organize microtubules. Elongating spermatids display gamma-tubulin spots in the neck region, while microtubules are organized from the perinuclear ring as the manchette. Electron microscopic studies using immunogold labeling revealed that gamma-tubulin is mainly localized in the centriolar adjunct from which an aster of microtubules emanates. Microtubules repolymerized randomly in the cytoplasm after nocodazole treatment and reversal. gamma-Tubulin dissociates from the neck region and is discarded in the residual bodies during spermiation. The distal centriole degenerates during testicular stage of spermiogenesis, while the proximal centriole is lost during epididymal stage. Loss of centrosomal protein and centrioles in mouse sperm further confirm the maternal inheritance of centrosome during murine fertilization.
Animals↗
The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components spc97p and spc98p.
gamma-Tubulin is a universal component of microtubule organizing centers where it is believed to play an important role in the nucleation of microtubule polymerization. gamma-Tubulin also exists as part of a cytoplasmic complex whose size and complexity varies in different organisms. To investigate the composition of the cytoplasmic gamma-tubulin complex in mammalian cells, cell lines stably expressing epitope-tagged versions of human gamma-tubulin were made. The epitope-tagged gamma-tubulins expressed in these cells localize to the centrosome and are incorporated into the cytoplasmic gamma-tubulin complex. Immunoprecipitation of this complex identifies at least seven proteins, with calculated molecular weights of 48, 71, 76, 100, 101, 128, and 211 kD. We have identified the 100- and 101-kD components of the gamma-tubulin complex as homologues of the yeast spindle pole body proteins Spc97p and Spc98p, and named the corresponding human proteins hGCP2 and hGCP3. Sequence analysis revealed that these proteins are not only related to their respective homologues, but are also related to each other. GCP2 and GCP3 colocalize with gamma-tubulin at the centrosome, cosediment with gamma-tubulin in sucrose gradients, and coimmunoprecipitate with gamma-tubulin, indicating that they are part of the gamma-tubulin complex. The conservation of a complex involving gamma-tubulin, GCP2, and GCP3 from yeast to mammals suggests that structurally diverse microtubule organizing centers such as the yeast spindle pole body and the animal centrosome share a common molecular mechanism for microtubule nucleation.
3T3 Cells↗
Parallel analysis of genetic selections using whole genome oligonucleotide arrays.
Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein-protein interaction screens were carried out for S. cerevisiae genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.
Animals↗
Expression of amino- and carboxyl-terminal gamma- and alpha-tubulin mutants in cultured epithelial cells.
Three distinct tubulin proteins are essential for microtubule function: alpha-, beta-, and gamma-tubulin. After translation, alpha- and beta-tubulin proteins combine into a soluble, 7 S heterodimer that is multimerized to form the microtubule filament. Conversely, gamma-tubulin combines with several proteins into a soluble, 25 S multi-protein particle, the gammasome that is essential for nucleating microtubule filaments at the centrosome. The proteins that assist tubulins in executing their specific functions are largely unknown. As an initial approach to address this issue, we first decided to identify domains of mammalian alpha- and gamma-tubulin necessary for their function by creating mutant mammalian alpha- and gamma-tubulin (both deletion and hybrid mutants) and assaying their behavior in stably transfected Chinese hamster ovary epithelial cells. First, we demonstrated that addition of a carboxyl-terminal epitope tag had no effect on the subcellular localization of either alpha- and gamma-tubulin. Second, we found that both the amino and carboxyl termini of gamma-tubulin were essential for its incorporation into the gammasome. Third, we found that the amino and carboxyl termini of alpha-tubulin were necessary for incorporation of the alpha-beta-tubulin heterodimer into the microtubule filament network. In general, alpha-tubulin sequences could not replace those of gamma-tubulin and vice versa. Taken together, these results suggest that the amino and carboxyl termini of alpha- and gamma-tubulin and perhaps regions throughout these proteins were necessary for their specific functions.
Amino Acid Sequence↗
Nucleation and capture of large cell surface-associated microtubule arrays that are not located near centrosomes in certain cochlear epithelial cells.
This report deals with the as yet undetermined issue of whether cell-surface associated microtubules in certain cochlear epithelial cells are centrosomally nucleated and subsequently migrate to microtubule-capturing sites located at the surface regions in question. Alternatively, the cells may possess additional nucleating sites which are noncentrosomal and surface-associated. These alternative possibilities have been investigated for highly polarised epithelial cells called supporting cells in the mouse and guinea pig organ of Corti using antibodies to pericentrin and gamma-tubulin. There is substantial evidence that both proteins are essential components of microtubule-nucleating sites in cells generally. Each mature supporting cell possesses a large microtubule array that is remotely located with respect to its centrosome (more than 10 microns away). The antibodies bind to a cell's centrosome. No binding has been detected at 2 other microtubule-organising centres that are associated with the ends of the centrosomally-remote microtubule array while it is being constructed. Such arrays include thousands of microtubules in some of the cell types that have been examined. If all a cell's microtubules are nucleated by its centrosome then the findings reported above imply that microtubules escape from the centrosomal nucleating site and migrate to a new location. Furthermore capture of the plus and minus ends of the errant microtubules is taking place because both ends of a centrosomally-remote microtubule array are attached to sites that are precisely positioned at certain cell surface locations. Minus ends are locating targets with an exactitude comparable to that which has been demonstrated for plus ends in certain cell types. These cells apparently operate a single control centre strategy for microtubule nucleation that is complemented by precise positioning of plus and minus end-capturing sites at the cell surface.
Animals↗
Cytoskeleton: anatomy of an organizing center.
One component of the yeast spindle pole body, Spc42p, has been found to form a crystalline array within one of the central layers of the structure; the Spc42p crystal might provide a scaffold around which the spindle pole body is assembled, and could be involved in regulating the size of the spindle pole body.
Centrosome↗