gammaTubulin at 23C

EVOLUTIONARY HOMOLOGS (part 1/2)

A second Drosophila gamma tubulin

A study was made of the mutant phenotypes brought about during early embryogenesis by mutation in the gammaTub37C gene, one of the two isoforms of gamma-tubulin that have been identified in Drosophila. Attention was focused on fs(2)TW1(1) and fs(2)TW1(RU34), null and hypomorph alleles, respectively, of this gene, whose sequences are reported in this paper. The abnormal meiotic figures observed in mutant stage 14 oocytes are not observed in laid oocytes or fertilized embryos, suggesting that these abnormal meiotic figures are not terminally arrested. Both null and hypomorph alleles lead to a total arrest of nuclear proliferation during early embryogenesis. This is in contrast to their effect on female meiosis-I, where hypomorph alleles display a much weaker phenotype. Null and hypomorph alleles lead to some distinct phenotypes. Unfertilized laid oocytes and fertilized embryos deficient for gammaTub37C do not contain polar bodies and have a few bipolar microtubule arrays. In contrast, oocytes and embryos from weaker alleles do not have these microtubule arrays, but do contain polar bodies, or polar-body-like structures. These results indicate that gammaTub37C is essential for nuclear proliferation in the early Drosophila embryo (Llamazares, 1999).

Gamma tubulin in yeast

The mipA gene of A. nidulans encodes gamma-tubulin. mipA 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 a virtually complete absence of mitotic apparatus. It is concluded 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. It is proposed that gamma-tubulin attaches microtubules to the spindle pole body, nucleates microtubule assembly, and establishes microtubule polarity in vivo (Oakley, 1990).

Gamma-Tubulin exists in fission yeast as the product of an essential gene, encoding a 446 amino acid protein that is 77.3% identical to Aspergillus nidulans gamma-tubulin. Gene disruption in yeast causes cell lethality, displaying condensed, undivided chromosomes with aberrant spindle structures. Anti-gamma-tubulin staining shows that gamma-tubulin is located, throughout the wild-type cell cycle, at the spindle pole bodies (SPBs), indicating that gamma-tubulin associates with interphase SPB in the absence of microtubules. In addition, anti-gamma-tubulin immunofluorescence staining reveals cytoplasmic, cell-equatorial putative MTOCs (microtubule organizing centers), which appear only during mitotic telophase and cytokinesis, and are located at the centers for the new cytoplasmic microtubule arrays of the two daughter cells. In the multiple-SPB mutant cut1-cdc11, anti-gamma-tubulin antibodies reveal many dots on the periphery of the nucleus. These results confirm that gamma-tubulin is an important member of the tubulin superfamily, and suggest that it may be a universal component of MTOCs, consistent with a role for gamma-tubulin in controlling microtubule formation in vivo (Horio, 1991).

The predicted amino acid sequence of the yeast Tub4 protein (Tub4p) is 29-38% identical to members of the gamma-tubulin family. Indirect immunofluorescence experiments using a strain containing an epitope-tagged Tub4p indicate that Tub4p resides at the spindle pole body (SPB) throughout the yeast cell cycle. Deletion of the TUB4 gene indicates that Tub4p is essential for yeast cell growth. Tub4p-depleted cells arrest during nuclear division; most arrested cells contain a large bud, replicated DNA, and a single nucleus. Immunofluorescence and nuclear staining experiments indicate that cells depleted of Tub4p contain defects in the organization of both cytoplasmic and nuclear microtubule arrays; such cells exhibit nuclear migration failure, defects in spindle formation, and/or aberrantly long cytoplasmic microtubule arrays. These data indicate that the S. cerevisiae gamma-tubulin protein is an important SPB component that organizes both cytoplasmic and nuclear microtubule arrays (Sobel, 1995).

Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle defects as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM reveals that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules (Marschall, 1996).

The essential Tub4p is associated with both the inner and outer plaques of the yeast microtubule organizing center, two substructures of the spindle pole body. These SPB substructures are involved in the attachment of the nuclear (to inner plaques) and cytoplasmic microtubules (to outer plaques). Study of a temperature sensitive tub4-1 allele reveals that TUB4 has essential functions in microtubule organization. Remarkably, SPB duplication and separation are not impaired in tub4-1 cells incubated at the nonpermissive temperature. However, SPBs from such cells contain less or misdirected nuclear microtubules. Further analysis revealed that tub4-1 cells are able to assemble a short bipolar spindle, suggesting that the defect in microtubule organization occurs after spindle formation. A role for Tub4p in microtubule organization is further suggested by an increase in chromosome loss in tub4-1 cells. In addition, cell cycle arrest and survival of tub4-1 cells is dependent on the mitotic checkpoint control gene BUB2, one of the cell's monitors of spindle integrity (Spang, 1996).

The yeast gamma-tubulin, Tub4p, forms a 6S complex with the spindle pole body components Spc98p and Spc97p. Purified Tub4p complex contains one molecule of Spc98p and Spc97p, and two or more molecules of Tub4p, but no other protein. Studies have been carried out to determine how the Tub4p complex binds to the yeast microtubule organizing center, the spindle pole body (SPB). Genetic and biochemical data indicate that Spc98p and Spc97p from the Tub4p complex bind to the N-terminal domain of the SPB component Spc110p. A complex has been isolated containing Spc110p, Spc42p, calmodulin and a 35 kDa protein, suggesting that these four proteins interact in the SPB. This paper describes how the N-terminus of Spc110p anchors the Tub4p complex to the SPB and how Spc110p itself is embedded in the SPB (Knop, 1997b).

The yeast microtubule organizing center (MTOC), known as the spindle pole body (SPB), organizes the nuclear and cytoplasmic microtubules, which are functionally and spatially distinct. The SPB of Saccharomyces cerevisiae offers an example of one MTOC that can organize functionally and spatially distinct classes of microtubules. The S. cerevisiae SPB is embedded in the nuclear envelope during the entire cell cycle. Substructures named the outer, central and inner plaques have been described by electron microscopy. The outer and inner plaques organize the cytoplasmic and nuclear microtubules, respectively. The cytoplasmic microtubules have functions in nuclear positioning and nuclear movement, while the nuclear microtubules are involved in spindle formation and chromosome segregation in mitosis and meiosis. The S. cerevisiae gamma-tubulin, Tub4p, forms a stable complex with two other proteins, Spc98p and Spc97p, and this gamma-tubulin complex (Tub4p complex) is localized at the outer and inner plaques of the SPB. Conditional lethal mutants in SPC98 and SPC97 reveal a role of the encoded proteins in microtubule organization by the SPB (Knop, 1998 and references).

Microtubule organization requires the Tub4p complex, which binds to the nuclear side of the SPB at the N-terminal domain of Spc110p. In this paper, the essential SPB component Spc72p is described, whose N-terminal domain interacts with the Tub4p complex on the cytoplasmic side of the SPB. This Tub4p complex-binding domain of Spc72p is essential: 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 accumulate in these cultures. In contrast, overexpression of the entire SPC72 results in more cytoplasmic microtubules, as compared with wild-type. Exchange of the Tub4p complex-binding domains of Spc110p and Spc72p establishes that the Spc110p domain, when attached to DeltaN-Spc72p, is functional at the cytoplasmic site of the SPB, while the corresponding domain of Spc72p, when fused to DeltaN-Spc110p, leads 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 (Knop, 1998).

The components of the yeast Tub4p complex have been localized to the outer and inner plaques, suggesting that they represent universal components of the microtubule organization machinery. In contrast, Spc110p and Spc72p are the first side-specific proteins of the SPB involved in microtubule organization. These data suggest that Spc110p and Spc72p have at least two functionally distinct domains: an N-terminal domain that interacts with the Tub4p complex and a C-terminal domain, which binds to at least one other SPB component. Cell-cycle-dependent modification of the N-terminal domain of Spc110p and Spc72p could in fact modify the microtubule organization properties of the inner and outer plaques. In this respect, it is interesting that Spc110p is a phosphoprotein and that Spc72p was resolved by SDS-PAGE into multiple bands, suggesting that it is subject to modification. The C-terminal domains of Spc110p and Spc72p carry the information as to which side of the SPB the proteins bind. Spc110p has been purified in complex with Spc42p, calmodulin and an SPB component with an apparent mol. wt of 35 kDa, indicating a physical interaction between the four SPB components. How Spc72p is bound to the SPB is still an open question (Knop, 1998 and references).

A trimeric complex formed by Tub4p, the budding yeast gamma-tubulin, and the two spindle pole body components, Spc98p and Spc97p, has recently been characterized in Saccharomyces cerevisiae. Primers derived from the human expressed sequence tag matching SPC98 were used to clone the 5' and 3' cDNA ends by rapid amplification of cDNA ends (RACE)-PCR. The human Spc98 cDNA presents an alternative splicing at the 3' end. The deduced protein possesses 22% identity and 45% similarity with the yeast homolog. The human Spc98p, like gamma-tubulin, is concentrated at the centrosome, although a large fraction is found in cytosolic complexes. Sucrose gradient sedimentation of the cytosolic fraction and immunoprecipitation experiments demonstrate that both gamma-tubulin and HsSpc98p are in the same complex. Interestingly, Xenopus sperm centrosomes, which are incompetent for microtubule nucleation before their activation in the egg cytoplasm, are found to contain similar amounts of both Spc98p and gamma-tubulin to human somatic centrosomes, which are competent for microtubule nucleation. Affinity-purified antibodies against Spc98p inhibit microtubule nucleation on isolated centrosomes, as well as in microinjected cells, suggesting that this novel protein is indeed required for the nucleation reaction (Tassin, 1998).

In budding yeast, microtubule organizing functions are provided by the spindle pole body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes two main sets of microtubules. The cytoplasmic outer plaque and the nuclear inner plaque are SPB substructures that are associated with the cytoplasmic and nuclear microtubules, respectively. An additional SPB substructure that functions in cytoplasmic microtubule (CM) organization is the half bridge, a one-sided extension of the central plaque layered on top of the nuclear envelope. Nuclear and cytoplasmic microtubules are attached to the inner and outer plaque of the SPB throughout the cell cycle. In contrast, CMs originate only in G1 of the cell cycle from the SPB half bridge. During vegetative growth the nuclear microtubules are involved in SPB separation, spindle formation and chromosome segregation, whereas the CMs function in SPB orientation, spindle positioning and in migration of the nucleus into the bud (Pereira, 1999).

Upon the exposure of yeast cells to mating pheromone, the CMs reorganize so that they are directed from the half bridge of the SPB towards the mating projection. This study addresses the question of how gamma-tubulin becomes attached to the SPB half bridge during the process of mating. Following microtubule reorganization, a and alpha cells fuse; the two nuclei are moved together along the interdigitating microtubules until they finally fuse, a step known as karyogamy. Genes involved in karyogamy have been identified by genetic screens. Kar1p is particularly interesting since it is found at the half bridge of the SPB and functions in both karyogamy and SPB duplication. However, little is known about the molecular role of Kar1p in karyogamy and its possible involvement in the pheromone-induced rearrangement of the CMs at the SPB (Pereira, 1999).

How microtubules form in vivo became clearer with the discovery of gamma-tubulin as a universal component of centrosomes and SPBs that is involved in microtubule formation. gamma-tubulin is found as part of a large complex containing gamma-tubulin, homologs of yeast Spc98p and Spc97p and additional proteins, depending on the organism. In budding yeast the gamma-tubulin or Tub4p complex consists of the gamma-tubulin, named Tub4p, and the SPB components Spc98p and Spc97p. Like other gamma-tubulin complexes, the Tub4p complex assembles in the cytoplasm and is then targeted to the sites of microtubule organization at the SPB via the binding of Spc97p and Spc98p to the N-terminal domain of Spc72p and of Spc110p. Spc72p is associated with the cytoplasmic outer plaque, whereas Spc110p is located with the nuclear inner plaque, explaining how the Tub4p complex is bound to the cytoplasmic and nuclear sides of the SPB. The term gamma-tubulin complex-binding protein (GTBP) has been coined for proteins such as Spc72p and Spc110p that target gamma-tubulin complexes to distinct cellular localizations (Pereira, 1999).

How Spc110p is embedded within the SPB is relatively well understood. Spc110p has been purified in a complex containing yeast calmodulin (Cmd1p), and the SPB components Spc29p and Spc42p. Spc42p forms a layer within the center of the SPB to which the C-terminus of Spc110p is anchored via Spc29p. Cmd1p binds to a site in the C-terminus of Spc110p, which is adjacent to the Spc29p-binding domain. Evidence has been obtained that Cmd1p binding to Spc110p may regulate the interaction of Spc110p with Spc29p. In contrast, the binding proteins of Spc72p at the SPB have not been identified. However, it is known that the C-terminus of Spc72p is sufficient to target this protein to the SPB. How the CMs of the half bridge are organized in G1 of the cell cycle and in mating cells is largely unknown. Evidence that the Tub4p complex-binding protein Spc72p interacts directly with the N-terminal domain of the half bridge component Kar1p, explains the function of Kar1p in conjugation. By means of this interaction the Tub4p complex is targeted to the half bridge where it organizes CMs. Evidence that the interaction of Kar1p with Spc72p has only a minor function during vegetative growth, but becomes essential for CM organization during conjugation. The gamma-tubulin complex-binding protein Spc72p interacts directly with the N-terminal domain of Kar1p, thereby targeting the gamma-tubulin complex to the half bridge. The localization of Spc72p within the spindle pole body changes throughout the cell cycle and even more strongly in response to mating pheromone. Taken together, these observations suggest that the relocalization of Spc72p within the spindle pole body is the 'landmark' event in the pheromone-induced reorganization of the cytoplasmic microtubules (Pereira, 1999).

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. gamma-Tubulin is essential for nucleation, and its role can now be extended to these latter processes. A mutation in gamma-tubulin has been characterized that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant gamma-tubulin is similar to that of the wild-type protein. Prediction of gamma-tubulin structure indicates that non-alpha/betatubulin protein-protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the gamma-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appears unaltered, consistent with a redundant function for this protein in wild type. These data indicate a broader role for gamma-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. It is proposed that Pkl1p rescues an impaired function of gamma-tubulin that involves non-tubulin protein-protein interactions, presumably with a second motor, MAP, or MTOC component (Paluh, 2000).

Members of the Ncdp/Kar3p, BimCp, and MCAKp kinesin-like protein (klp) families can affect centrosome integrity, microtubule number and length, and/or the balance of forces for spindle assembly and function. In addition, Ncdp has been shown to be required for proper localization of gamma-tubulin to the meiosis II spindle. The Schizosaccharomyces pombe klp Pkl1p localizes to the SPB and spindle. It is nonessential in wild type; however, moderate overexpression of Pkl1p results in spindle shrinkage or collapse. To identify functional overlap with Pkl1p, mutations conferring dependence on pkl1 for viability were isolated. Two mutants recovered suggest that Pkl1p and a subset of gamma-tubulin functions are closely linked. One of these mutants is an allele of gamma-tubulin. Mutation of a single conserved residue allows microtubule nucleation but impairs chromosome segregation and has dramatic effects on cytoplasmic microtubule arrays. This analysis identifies a novel role for gamma-tubulin at the MTOC in regulating microtubule organization and dynamics (Paluh, 2000).

The essential process impaired in the gamma-tubulin PL301 mutant is the ability to effectively segregate chromosomes. A bipolar spindle is formed, and chromosomes are condensed and grouped, but anaphase A is blocked. The spindle checkpoint protein Mad2p (Drosophila homolog: CG17498) senses both attachment and tension of kinetochore microtubules, and Mad2p removal occurs as kinetochore microtubules accumulate. In S. pombe, as in mammalian cells, multiple microtubules attach to each kinetochore, and Mad2p regulates the spindle checkpoint. The extent of kinetochore microtubule attachment to chromosomes in the mutant at 19°C is unclear. At elevated temperatures segregation does occur; however, trailing chromosomes in many cells indicate that chromosome attachment is not always effectively maintained. Deletion of the spindle checkpoint gene mad2 removes the cold-sensitive mitotic arrest without severely reducing cell viability. This favors the view that microtubule attachment to kinetochores is incomplete or lacks tension, rather than being altogether absent. In budding yeast S. cerevisiae, a variety of lesions, including spindle pole-associated events, are able to activate the spindle checkpoint. It is possible in the mutant that Mad2p, in addition to sensing problems at the kinetochore, is also detecting a problem at the spindle poles relating to the gamma-tubulin functions that have been proposed here (Paluh, 2000).

The failure of appropriate kinetochore attachment is attributed to the increased microtubule stability and impaired dynamics in the mutant. The fact that cold temperatures exacerbate the phenotype is consistent with previous studies demonstrating that two classes of microtubules are especially stable at cold temperatures, kinetochore microtubules and microtubules of the midbody. Stabilization of spindle microtubules against depolymerization could prevent effective search and capture of kinetochores and block timely removal of the spindle and may have contributed to spindle hyperelongation (Paluh, 2000).

Growth at 30°C does not result in mitotic arrest of gtb1-PL301 cells. However, without multicopy pkl1 present, cytoplasmic microtubule arrays are combined into a few bundles or a single large bundle. 3D time-lapse video microscopy using GFP-alpha-tubulin shows that a single microtubule or a subset of microtubules are often extruded briefly, a short distance from the large bundle end. Extruded microtubules are dynamic and undergo growth and catastrophe, but the length of the large bundle itself is maintained or increased in size. This suggests that microtubule polymerization is outpacing depolymerization (Paluh, 2000).

In S. pombe, new cell equatorial MTOCs become active for nucleation late in mitosis and along with SPB MTOCs contribute to the microtubule arrays of daughter cells. Multicopy pkl1 is not able to alleviate mitotic arrest in the mutant; however, it does restore normal cytoplasmic microtubule arrays and cell morphology. Because there is no evidence that Pkl1p has cytoplasmic functions, it is expected that excess Pkl1p may improve the transition in microtubule arrays from mitosis to cytokinesis, perhaps by helping remove the spindle. In the mutant, retention of the spindle during cytokinesis results in a prominent knot of microtubules that impairs formation of these arrays. Abnormal astral microtubules may have contributed to the spindle hyperelongation phenotype that was also observed. The extended length or absence of astral microtubules may interfer with any cortical sensing of spindle length for triggering depolymerization of spindle microtubules (Paluh, 2000).

The gamma-tubulin mutation gtb1-PL301 is synthetically lethal with loss of Pkl1p. Another mutant isolated in the same screen, SL2, is suppressible by multicopy gamma-tubulin and arrests with defects in chromosome segregation. Both mutations suggest that Pkl1p function may be closely linked with that of gamma-tubulin in regulating microtubule dynamics for anaphase A. Another member of this family of klps, Drosophila Ncd, is required for proper localization of gamma-tubulin during meiosis II in vivo (Paluh, 2000).

Pkl1p may or may not interact directly with gamma-tubulin to rescue the mutant phenotype. However, a mechanism requiring Pkl1p binding to gamma-tubulin is favored, based on the localization of Pkl1p, involvement of gamma-tubulin with SL2, and preliminary evidence that Pkl1p antibody can coimmunoprecipitate gamma-tubulin. The localization of Pkl1p and its ability to affect spindle dynamics appear normal in the gamma-tubulin mutant. Thus, if Pkl1 binds to gamma-tubulin, it may rescue or restore an altered protein interaction at another site on gamma-tubulin, closer to the site of the mutation. This view makes sense because Pkl1p is not essential in wild type, and impaired Pkl1p function should be tolerated. Because multicopy pkl1 allows normal cytoplasmic arrays, the mutation is unlikely to enhance Pkl1p binding or function. If Pkl1p does bind to gamma-tubulin, this interaction could help tether or organize microtubules at the MTOC, because Pkl1p can bundle microtubules in vitro. Alternatively, such an interaction could be required for tubulin dimer removal by Pkl1p. It is not known whether yeast kinetochore microtubules undergo flux and, if so, what contribution it has to anaphase A. Although the kinetochore is the primary site of microtubule disassembly in somatic cells for anaphase A, flux coupled to depolymerization of microtubule minus ends is the predominant mechanism in Xenopus. Further investigation will be needed to decipher the exact mechanism underlying Pkl1p rescue of this subset of gamma-tubulin functions (Paluh, 2000).

The PL301 mutation is not positioned near known sites of tubulin-tubulin contact and so is not expected to affect microtubules directly. It lies at the edge of a divergent region on the gamma-tubulin surface, which is flanked on the opposite side by two helices. The similarly positioned helices in microtubules, helices 11 and 12, have been identified as primary sites of interaction with klp motor domains. The importance of a divergent face on gamma-tubulin is unknown. Still, the 3D model is expected to provide a useful tool for analyzing other gamma-tubulin mutations as well as non-tubulin protein interactions (Paluh, 2000).

Two obvious candidates for proteins whose functions may be altered in gtb1-PL301 are the conserved gamma-tubulin binding MTOC components. Sequence homologs to the S. cerevisiae Spc97p and Spc98p proteins are present in the S. pombe database. Another protein that helps organize spindle poles in metazoans and astral microtubules in fungi is dynein. How dynein interacts with microtubules or other proteins to accomplish this is not known. Although loss of dynein is synthetically lethal with loss of Kar3p in S. cerevisiae or loss of BimCp in A. nidulans, two klps with known mitotic functions, it is not synthetically lethal with loss of Pkl1p. However, it is still possible that dynein may influence organization of astral or interphase arrays in S. pombe (Paluh, 2000).

The ability of gamma-tubulin to associate with specialized classes of proteins, including MTOC components, microtubules, and motor proteins, is practical. It would provide this central MTOC component with the capability to intimately regulate microtubule minus end dynamics and organization as well as nucleation. These results clearly indicate that gamma-tubulin itself can dramatically affect microtubule dynamics and organization. With this broader view of gamma-tubulin function, the challenge will be to decipher the protein interactions that mediate both important roles and how these interactions are coordinated (Paluh, 2000).

gamma-Tubulin was originally identified as an extragenic suppressor of a mutation in beta-tubulin, A. nidulans benA33, that resulted in mitotic arrest with a stable bipolar spindle. Its discovery in this manner has remained intriguing, because spindle nucleation obviously occurred and because alpha-tubulin is now known to be unequivocally positioned as the ultimate subunit at the minus end of the microtubule. Therefore, an end-on interaction between gamma-tubulin and beta-tubulin is unlikely. It has been postulated that the role of gamma-tubulin in suppression of the benA33 phenotype might not relate to microtubule nucleation but instead to some as yet undefined role for gamma-tubulin. Although the mechanism of gamma-tubulin suppression of benA33 mitotic arrest is unknown, striking similarities are present with the gtb1-PL301 phenotype. Both results clearly argue for a broader view of gamma-tubulin function (Paluh, 2000).

Microtubule polymerization is initiated from the microtubule organizing center (MTOC), which contains the gamma-tubulin complex. This paper describes the identification of fission yeast Alp4 and Alp6, which are homologs of the gamma-tubulin-interacting proteins Sc.Spc97/Hs.Gcp2 and Sc.Spc98/Hs.Gcp3, respectively. The size of the fission yeast gamma-tubulin complex is large (>2000 kDa), comparable to that in metazoans. Both Alp4 and Alp6 localize to the spindle pole body (SPB) and also to the equatorial MTOC. Temperature-sensitive (ts) alp4 and alp6 mutants show two types of microtubular defects: (1) monopolar mitotic spindles form; (2) abnormally long cytoplasmic microtubules appear that do not stop at the cell tips and are still associated with the SPB. Alp4 function is required in G1 phase and ts mutants become lethal before S-phase. alp4 and alp6 mutants are hypersensitive to the microtubule-destabilizing drug thiabendazole (TBZ) and show a lethal 'cut' phenotype in its presence. Furthermore, alp4mad2 double mutants show an exaggerated multiple septation phenotype in TBZ. These results indicate that Alp4 and Alp6 may play a crucial role in the spindle pole-mediated checkpoint pathway (Vardy, 2000).

In wild type, cytoplasmic microtubules exist as several distinct filaments, most of which do not associate with the SPB. In contrast, alp4 mutant cells exhibit the following two abnormalities: (1) one or two greatly elongated microtubule bundles are seen; (2) these long microtubules generally associate with or pass through the SPB. The localization of the end marker Tea1 is not abolished in the alp4 mutant. This suggests that the longer cytoplasmic microtubules may arise due to defects in microtubule organization at the SPB rather than a failure in Tea1-dependent growth polarity control (Vardy, 2000).

It is possible that in the alp4 mutant, by interacting with the SPB, these interphase microtubules become more stabilized than wild-type forms. Thus, Alp4 and Alp6 may play an important role in both the maintenance of the length and the orientation of cytoplasmic microtubules. A similar phenotype displaying longer microtubules has been observed in mutations in the gamma-tubulin gene in budding yeast and recently in fission yeast. Defective phenotypes arising from ectopic overproduction of Alp4 appear similar to the mutant in terms of interphase microtubule morphology. This supports a role for these proteins in the maintenance of interphase microtubule integrity (Vardy, 2000).

It remains to be determined how interphase microtubules are generated and organized. It is possible that they originate at the MTOC (SPB and/or the equatorial MTOC) and are then released into the cytoplasm. Alternatively, the nucleation of cytoplasmic microtubules could be independent of the MTOC, but still require some interaction with it. Alp4 and Alp6 may be necessary for the liberation of cytoplasmic microtubules from the MTOC by some biochemical processes such as microtubule-severing reactions. Microtubule-nucleating activity is under cell cycle control in fission yeast, and it is now important to address the molecular mechanisms underlying cell cycle-dependent regulation of MTOC function (Vardy, 2000).

Alp4 and Alp6 are involved in the spindle assembly checkpoint pathways. alp4 and alp6 mutants do not activate the Mad2-dependent checkpoint as other mitotic mutants do (e.g. mutations in ß-tubulin encoding nda3 or kinesin-encoding cut7). Instead, Alp4 and Alp6 are required for the activation of the spindle checkpoint pathway per se. This is a unique character because, in addition to checkpoint control, Alp4 and Alp6 play an essential role as structural components involved in microtubule/spindle biogenesis. The reason why alp4 mutants do not activate the Mad2 pathway might be attributable to the location of Alp4 and Alp6. Analysis from several organisms suggests that the Mad2 pathway monitors the state of kinetochores or kinetochore-spindle interactions. Because Alp4 and Alp6 localize to the SPB rather than the kinetochores, kinetochore-spindle interactions might not be compromised, resulting in a failure to activate the Mad2-dependent checkpoint (Vardy, 2000).

It has become clear that spindle assembly checkpoint control bifurcates, and both kinetochore- and the SPB-mediated pathways exist. The kinetochore checkpoint is believed to arrest the cells in metaphase by preventing sister chromatid segregation, while the SPB-mediated pathway is involved in the inhibition of mitotic exit, which in fission yeast involves septation and cytokinesis. The fact that the alp4mad2 double mutant causes an exaggeration of the TBZ-sensitive phenotype at 26°C suggests that these two proteins may function independently in their response to this drug. Given the localization of Alp4 and Alp6 to the SPB, it is possible that Alp4 is involved in the SPB-mediated pathway. Unlike the kinetochore-mediated pathway, in which most of the components are conserved from yeasts to humans, components involved in the SPB checkpoint pathway so far have only been identified in yeast (e.g. the aforementioned Cdc16/Bub2, Byr4/Bfa1, Spg1/Tem1 and Cdc7/Cdc15). Given the universal conservation of the gamma-tubulin complex, it is proposed that a centrosome- and gamma-tubulin complex-dependent checkpoint is operational in vertebrates, in which Gcp2 and Gcp3 may play a crucial role (Vardy, 2000).

gamma-Tubulin is a conserved essential protein required for assembly and function of the mitotic spindle in humans and yeast. For example, human gamma-tubulin can replace the gamma-tubulin gene in Schizosaccharomyces pombe. To understand the structural/functional domains of gamma-tubulin, a systematic alanine-scanning mutagenesis of human gamma-tubulin (TUBG1) was performed and phenotypes of each mutant allele were examined in S. pombe. This screen, both in the presence and absence of the endogenous S. pombe gamma-tubulin, resulted in 11 lethal mutations and 12 cold-sensitive mutations. Based on structural mapping onto a homology model of human gamma-tubulin generated by free energy minimization, all deleterious mutations were found in residues predicted to be located on the surface, some in positions to interact with alpha- and/or beta-tubulins in the microtubule lattice. As expected, one class of tubg1 mutations has either an abnormal assembly or loss of the mitotic spindle. Surprisingly, a subset of mutants with abnormal spindles does not arrest in M phase but proceeds through anaphase followed by abnormal cytokinesis. These studies reveal that in addition to its previously appreciated role in spindle microtubule nucleation, gamma-tubulin is involved in the coordination of postmetaphase events, anaphase, and cytokinesis (Hendrickson, 2001).

The budding yeast gamma-tubulin (Tub4p) is phosphorylated in vivo. Hyperphosphorylated Tub4p isoforms are restricted to G1. A conserved tyrosine near the carboxy terminus (Tyr445) is required for phosphorylation in vivo. A point mutation, Tyr445 to Asp, causes cells to arrest prior to anaphase. The frequency of new microtubules appearing in the SPB region and the number of microtubules are increased in tub4-Y445D cells, suggesting this mutation promotes microtubule assembly. These data suggest that modification of gamma-tubulin is important for controlling microtubule number, thereby influencing microtubule organization and function during the yeast cell cycle (Vogel, 2001).

What is the role of Tub4p phosphorylation? Tub4p phosphorylation appears to regulate microtubule assembly, and thereby affects microtubule number, stability, and organization. In support of this hypothesis, it is found that the frequency of new microtubules detected at the SPBs and the number of astral microtubules are increased in the tub4-Y445D mutant, which mimics constitutive phosphorylation. Phosphorylation of Tub4p during G1 is likely to be important for stimulating microtubule assembly during spindle pole duplication. This would lead to the formation of a full complement of microtubules from each pole as cells prepare for pole separation and mitosis. The tub4-Y445D mutation has a more pronounced effect on microtubule assembly and stability than the opposing tub4-Y445F mutation. Although the reasons for this are not known, it is speculated that (1) phosphorylation of several Tub4p residues participates in promoting microtubule assembly in G1 (this is consistent with the detection of multiple acidic Tub4p isoforms in G1) and/or (2) dephosphorylation of Tub4p is critical for cell cycle progression. Dephosphorylation of Tub4p is presumably critical for mitosis (Vogel, 2001).

The organization of the core proteins of the yeast spindle pole body

The spindle pole body (SPB) is the microtubule organizing center of Saccharomyces cerevisiae. Its core includes the proteins Spc42, Spc110 (kendrin/pericentrin ortholog), calmodulin (Cmd1), Spc29, and Cnm67. Each was tagged with CFP and YFP and their proximity to one another was determined by fluorescence resonance energy transfer (FRET). FRET was measured by a new metric that accurately reflected the relative extent of energy transfer. The FRET values established the topology of the core proteins within the architecture of SPB. The N-termini of Spc42 and Spc29, and the C-termini of all the core proteins face the gap between the IL2 layer and the central plaque. Spc110 traverses the central plaque and Cnm67 spans the IL2 layer. Spc42 is a central component of the central plaque where its N-terminus is closely associated with the C-termini of Spc29, Cmd1, and Spc110. When the donor-acceptor pairs were ordered into five broad categories of increasing FRET, the ranking of the pairs specified a unique geometry for the positions of the core proteins, as shown by a mathematical proof. The geometry was integrated with prior cryoelectron tomography to create a model of the interwoven network of proteins within the central plaque. One prediction of the model, the dimerization of the calmodulin-binding domains of Spc110, was confirmed by in vitro analysis (Muller, 2005).

The spindle pole body is the microtubule organizing center of Saccharomyces cerevisiae (Jaspersen, 2004). Two SPBs establish the bipolar mitotic spindle, a defining event of mitosis that allows the stable transmission of equivalent genetic material to the mother and daughter cell at the time of cell division. This role of the SPB is carried out by the centrosome in higher eukaryotes (Muller, 2005).

The structure of the SPB is reviewed by Jaspersen (2004). Briefly the ultrastructure observed by electron tomography consists of a series of stacked layers embedded in the nuclear envelope. The inner plaque is the area where the microtubules dock to the SPB; this plaque harbors the gamma-tubulin complex and the N-terminus of Spc110. The central plaque and the IL2 layer are the two core layers. This core is composed of 5 proteins. Spc29 and Cmd1 reside in the central plaque. Spc42 is thought to begin within the central plaque, but terminate in the IL2 layer. The C-terminus of Spc110 is in the central plaque where it binds Cmd1. The C-terminus of Cnm67 lies in the IL2 layer where it binds Spc42 and links the SPB core to the outer plaque. The outer plaque is the cytoplasmic boundary of the SPB where the astral microtubules nucleate from a second region of gamma-tubulin. Based on primarily two-hybrid interactions the SPB core proteins are typically depicted as components lying along a linear path that proceeds from Spc110 to Spc29 to Spc42 to Cnm67 (Muller, 2005).

The ultrastructure of the SPB is clearly quite different from the centrosome. Centrioles are not present and the SPB remains inserted in the nuclear envelope during mitosis. Yet both have in common the gamma-tubulin complex, Spc110/kendrin/AKAP-450, calmodulin, centrin, and Sfi1p. (The latter two proteins are part of the SPB half-bridge, a domain involved in SPB duplication. Despite differences in gross anatomy, the SPB and centrosome likely share an underlying structure. To date the only component of either the SPB or centrosome whose structure is solved at atomic resolution is calmodulin. The paucity of structural information has limited the understanding of the molecular functions performed by individual SPB proteins. Without crystals or well behaved soluble proteins, the available research tools to probe the SPB structure or any large macromolecular complex are few (Muller, 2005).

This study used a hybrid approach that combined in vivo live-cell FRET measurements with previous cryo-EM analysis. CFP and YFP were used as FRET donor and acceptor and attached to the components of the SPB. Initially FRET values were classified as either positive or negative for energy transfer as judged by a comparison to carefully designed controls. This binary classification system allowed mapping of the ends of proteins within the architecture of the SPB. Next the positive values were subdivided into classes. The classification specified a unique geometry for the SPB components that was not only consistent with previous structural and genetic studies, but broadened the understanding of SPB organization (Muller, 2005).

The FRET results suggest that the Il2 layer and central plaque form an integrated meshwork of proteins with Spc42 closely associated with all components of the central plaque. The general features of the core proteins of the IL2 and central plaque, based on the current results and the general literature, are as follows. The N-terminus of Spc42 begins at the inner boundary of the central plaque, forms a coiled-coil domain that defines the spacing of the gap between layers, enters the IL2 layer, and finally loops back to end at the internal face of the IL2 layer. Remarkably, even though the N-terminal domain before the coiled coil is only ~60 amino acids long, the N-terminus is in close proximity to the C-termini of Spc29, Cmd1, and Spc110. Cnm67 begins at the outer plaque, penetrates the IL2 and ends in close proximity to the C-terminus of Spc42. The N- and C-termini of Spc29 both lie on the inner face of the central plaque. Cmd1 is situated near the C-terminal end of Spc110, consistent with in vitro binding experiments, genetic and two-hybrid results. Finally Spc110, which at its N-terminus binds the gamma-tubulin complex extends from the inner plaque through the central plaque and ends in close juxtaposition to the C-terminus of Spc42. All the termini of the central plaque and IL2 layer proteins lie along the internal edges of the IL2 and central plaque layers, facing the space between the two layers (Muller, 2005).

The SPB is organized around a hexagonal lattice of Spc42. The arrangement of Spc42 in the Il2 layer was suggested by analysis of cryoelectron micrographs of both SPB cores and two-dimensional crystals of Spc42 that arise in vivo upon Spc42 overexpression. Because the N-terminus of Spc42 is situated in the central plaque, the arrangement of Spc42 in the IL2 layer necessarily imposes the same organization on the location of Spc42 in the central plaque. Cryoelectron microscopy has not revealed this implied organization of the central plaque. However the visualization of the Spc42 arrangement in the IL2 relied upon the contrast between regions of high protein density and pockets of low or no density. If, as supported by the FRET results, the components of the central plaque are densely packed, a uniform and high protein density would mask the organization in electron micrographs (Muller, 2005).

The Spc42 lattice provided a template that enabled the FRET-based geometry of the core proteins to be taken and and a model to be generated for the organization of the central plaque. The model suggests that Spc42 and Spc29 form the heart of the central plaque. A strong association between Spc29 and Spc42 is well documented. Spc29 has a robust two-hybrid interaction with the N-terminus of Spc42. In an Spc110-226 mutant, Spc29 remains associated with Spc42 under the conditions in which Spc110-226, calmodulin, and the gamma-tubulin complex pull away from the SPB. Finally, Spc29 is seen with Spc42 at the satellite of the SPB. In this model Spc29 lies along the path of Spc42 and together they form a ring of protein around the center of the hexagonal unit in the central plaque (Muller, 2005).

At the center of the hexagonal unit is placed a trimer of Spc110 dimers as they unravel from their coiled coil motif. In the model Spc110 enters the central plaque through the ring of Spc29 and Spc42. Two-hybrid analysis suggested that Spc29 binds to Spc110 between the end of the coiled coil and the start of the Cmd1-binding domain, from positions 811 to 898. This region overlaps Region II of Spc110 (position 772-836), a domain that plays a role in locking Spc110 in place during mitosis. The FRET model is consistent with Spc29 and Spc42 acting as a clasp to surround and lock Spc110 in place. However the central plaque must not only lock Spc110 in place to withstand the push and pull of mitosis, but also must be organized in a way that facilitates the remodeling of the SPB during G1/S-phase when 50% of Spc110 turns over. Therefore any locking mechanism must be reversible and the interaction between Spc110 and Spc29 must be dynamic (Muller, 2005).

Calmodulin and the C-terminal domain of Spc110 are positioned to reinforce lateral stability of the central plaque. This is evident when the hexagonal unit is tessellated to form a mosaic lattice of the central plaque components. Calmodulin and the C-terminal domain of Spc110 from one hexagonal unit are juxtaposed with their counterparts in the adjoining hexagonal units. The dimerization of the C-terminal Spc110/Cmd1 domain was confirmed in vitro. Surprisingly even though calmodulin is a highly conserved component of the SPB, it is not required. An SPC110-407 mutant of S. cerevisiae that lacks the calmodulin-binding domain is still viable. One explanation is that the integrity of the SPB is maintained through structurally redundant lateral connections in IL2 layer and central plaque (Muller, 2005).

The tessellation of the repeat unit prompts the question of what determines the lateral limits of the SPB. How is the repeat symmetry broken and the boundary with the nuclear envelope established? One clue may come from a comparison of the dimensions of the SPB with the cluster of nuclear microtubules that originate at the SPB. The SPB is circular with an average diameter of ~165 nm for the central plaque from a diploid and therefore an area of ~2.1 x 10⁶Ų. A diploid would have ~35 microtubules emanating from the SPB (32 kinetochore microtubules and a three pole-to-pole microtubules. Microtubules have a cross-sectional diameter of 25 nm, so the minimal total area occupied by 35 microtubules (hexagonal packing with a packing density of 91% is 1.9 x 10⁶Ų. Even assuming some spread at the inner plaque, the SPB has almost the minimal area required to attach the nuclear microtubules. One mechanism that could minimize both the size of the SPB and the size of the bundle of microtubules would be feedback between microtubule attachment and Spc110 turnover. A removal of Spc110 molecules that are not nucleating microtubules would break the lattice symmetry, leaving Spc42 and Spc29 to interact with other proteins of the nuclear envelope. Spc110 is only added to the SPB after the insertion of Spc42 and Spc29 into the nuclear envelope, so the edge of the SPB does not require Spc110. The mechanism and role of Spc110 turnover is an area of continued research (Muller, 2005).

Gamma tubulin in Paramecium

First discovered in the fungus Aspergillus nidulans, gamma-tubulin is a ubiquitous component of microtubule organizing centers. In centrosomes, gamma-tubulin has been immunolocalized at the pericentriolar material, suggesting a role in cytoplasmic microtubule nucleation, as well as within the centriole core itself. Although its function in the nucleation of the mitotic spindle and of cytoplasmic interphasic microtubules has been demonstrated in vitro and in vivo, the hypothesis that gamma-tubulin could intervene in centriole assembly has never been experimentally addressed because the mitotic arrest caused by the inactivation of gamma-tubulin in vivo precludes any further phenotypic analysis of putative centriole defects. The issue can be addressed in the ciliate Paramecium, which are characterized by numerous basal bodies that are similar to centrioles but the biogenesis of these basal bodies is not tightly coupled to the nuclear division cycle. The inactivation of the Paramecium gamma-tubulin genes leads to inhibition of basal body duplication (Ruiz, 1999).

Gamma tubulin in C. elegans

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. As in other organisms, Caenorhabditis elegans gamma-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, transgenic worms were generated that express GFP::gamma-tubulin or GFP::ß-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms reveal 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, RNA interference (RNAi) was used to deplete C. elegans embryos of gamma-tubulin. gamma-Tubulin (RNAi) embryos fail in chromosome segregation, but surprisingly, they contain extensive microtubule arrays. Moderately affected embryos contain bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contain collapsed spindles with numerous long astral microtubules. These 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 (Strome, 2001).

It is interesting that microtubules emanating from gamma-tubulin-depleted centrosomes in C. elegans embryos are sufficient for some processes but not for others. For example, slow migration of the female pronucleus occurs, but fast pronuclear migration is inhibited. Both phases of pronuclear migration depend on microtubules. However, slow migration may depend on the meshwork of interphase microtubules, whereas directed fast migration may use microtubules emanating from the centrosomes of the male pronucleus. When sperm-aster microtubule plus ends contact minus-end-directed microtubule motors on the female pronucleus, the motors could then pull the pronuclei directly together. The inhibition of fast migration suggests that gamma-tubulin is important for the generation or function of sperm-aster microtubules that can support such force production (Strome, 2001).

Depletion of gamma-tubulin also appears to affect the functions of a subset of mitotic spindle microtubules. Duplicated centrosomes in severe gamma-tubulin(RNAi) embryos were capable of generating astral arrays of microtubules, but the asters were not properly separated, resulting in collapsed spindles. This suggests that gamma-tubulin is needed for stable interpolar connections, which are normally established by lateral interactions of microtubule plus ends from opposite poles. Spindles with separated poles often did form in less severely affected gamma-tubulin(RNAi) embryos. In these cases, chromosome congression to the metaphase plate and anaphase chromosome segregation fail, suggesting that the accumulation of stable kinetochore microtubules requires gamma-tubulin. Combining these results with observations that mitotic spindles without functional centrosomes can mediate normal chromosome segregation, it appears that the stabilizing influence of gamma-tubulin does not require that it reside in a centrosome. At least, it seems evident that gamma-tubulin function goes beyond the simple nucleation of microtubules (Strome, 2001).

The dynamics of C. elegans centrosomes in 1-cell embryos seen with rhodamine-alpha/ß-tubulin have been described. Observations of the dynamic behavior of GFP::gamma- and GFP::ß-tubulin confirm their results and provide additional insights. In particular, it has been documented that first ß-tubulin and then gamma-tubulin appear to shift from even distributions throughout the centrosome to expanding hollow spheres. ß-Tubulin shows the hollow distribution as early as prometaphase, whereas gamma-tubulin shows hollowing later, during anaphase. With both GFP fusion proteins, daughter centrosomes are seen within the mother spheres during anaphase. Depletion of gamma-tubulin reduces or eliminates the centrosome hollowing and expansion seen with GFP::ß-tubulin. This suggests that the alternative alpha/ß recruitment-nucleation mechanism is distributed evenly throughout the centrosome and cannot support the forces that cause outward centrosome expansion during anaphase (Strome, 2001).

It is interesting that the anterior and posterior mother centrosomes behave differently, especially in light of the importance of first-division asymmetries in establishing developmental lineages. Most of anaphase B spindle elongation is accomplished by movements of the posterior centrosome. It swings from side to side as it moves toward the posterior end of the embryo. After swinging, the posterior ring of gamma- and ß-tubulin flattens and appears to be pulled apart laterally. The anterior sphere does not flatten. Perhaps the forces that generate the posterior pole's swinging motions, its flattening, and its lateral dispersion are all generated by minus-end-directed microtubule motors concentrated at a few sites in the posterior-lateral cortex. When an astral microtubule plus end from the posterior centrosome is first captured by one of those sites, the spindle pole is drawn toward it, in a lateral and slightly posterior direction. Subsequent microtubule contacts with sites on the opposite side swing the centrosome back and further toward the posterior. Eventually, multiple sites are engaged on both sides; the centrosome is held in balance between them near the posterior end; and continued force production stretches the centrosome apart laterally. In support of the existence of sites in the posterior cortex that 'pull' astral microtubules toward them, it has been observed that, after removal of the spindle midzone, the posterior spindle pole moved rapidly and with transverse oscillations to the posterior cortex (Strome, 2001).

gamma-Tubulin functions in the nucleation of a discrete subset of microtubules in the eukaryotic flagellum

gamma-Tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of gamma-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented, its role in microtubule nucleation in the eukaryotic flagellum is unclear. Trypanosoma brucei has been used to investigate possible functions of gamma-tubulin in the formation of the 9 + 2 flagellum axoneme. T. brucei possesses a single flagellum and forms a new flagellum during each cell cycle. An inducible RNA interference (RNAi) approach was used to ablate expression of gamma-tubulin, and, after induction, it was observed that the new flagellum is still formed but is paralyzed, while the old flagellum is unaffected. Electron microscopy reveals that the paralyzed flagellum lacks central pair microtubules but that the outer doublet microtubules are formed correctly. These differences in microtubule nucleation mechanisms during flagellum growth provide insights into spatial and temporal regulation of gamma-tubulin-dependent processes within cells and explanations for the organization and evolution of axonemal structures such as the 9 + 0 axonemes of sensory cells and primary cilia (McKean, 2003).

Although it has long been accepted that basal bodies act as the microtubule organizing centers for eukaryotic flagella/cilia, the molecular mechanisms leading to axonemal microtubule nucleation are not well understood. The results presented here show a clear distinction between nucleation of central pair microtubules (a gamma-tubulin-dependent process) and outer doublet microtubules (a gamma-tubulin-independent process). The latter appears to require only the pre-existing template of the basal body/centriole triplet microtubules. Examination of the known structures within the basal body suggests that the transition zone is key to the explanation. Central pair nucleation occurs from a central structure at the distal end of the transition zone. It is suggested that this central structure serves to anchor two gamma-tubulin complexes capable of nucleating the two central axonemal microtubules. Indeed, gamma-tubulin has been localized precisely to this position in the basal body transition zone in the green algae Chlamydomonas, and T. brucei gamma-tubulin has been localized to the basal body (McKean, 2003 and references therein).

Specialized cilia are now recognized as providing sensory functions in a range of metazoans, and these cilia usually possess axonemes with a 9 + 0 configuration. The 9 + 0 axoneme is a characteristic feature of the primary cilium formed as an extension of the mature centriole in many cells within the mammalian body and in cells in culture in the G1/G0 phase of the cell cycle. There is recent evidence that the primary cilium exhibits specific receptors, and malfunction of this cilium leads to disease in mammals. The 9 + 0 axoneme is also found in sensory cells of invertebrates and in the modified sensory cilia of vertebrate photoreceptors. The distinction between central pair and outer doublet nucleation described here provides opportunities for differential regulation of microtubule extension into a 9 + 2 or a 9 + 0 axoneme. It appears likely that, during the normal proliferative cell cycle in mammalian cells or certain sensory cell differentiations, the gamma-tubulin-containing distal transition zone central structure is not formed on the mature centriole. Thus, the cell forms a 9 + 0 cilium that operates as a specific sensory organelle within the differentiated cell. Finally, defective assembly of axonemal central pair microtubules has also been observed in the motile cilia and sperm of individuals with diseases such as primary cilia dyskinesia (PCD). Given that central pair nucleation is dependent upon the construction of a gamma-tubulin complex within the distal central transition zone, identification of its molecular components may provide candidate genes for some of these primary ciliary dyskinesias (McKean, 2003).

Gamma tubulin in other insects

A longstanding enigma has been the origin of maternal centrosomes that facilitate parthenogenetic development in Hymenopteran insects. In young embryos, hundreds of microtubule-organizing centers (MTOCs) are assembled completely from maternal components. Two of these MTOCs join the female pronucleus to set up the first mitotic spindle in unfertilized embryos and drive their development. These MTOCs appear to be canonical centrosomes because they contain gamma-tubulin, CP190, and centrioles and they undergo duplication. Evidence is presented that these centrosomes originate from accessory nuclei (AN), organelles derived from the oocyte nuclear envelope. In the parasitic wasps Nasonia vitripennis and Muscidifurax uniraptor, the position and number of AN in mature oocytes correspond to the position and number of maternal centrosomes in early embryos. These AN also contain high concentrations of gamma-tubulin. In the honeybee, Apis mellifera, distinct gamma-tubulin foci are present in each AN. Additionally, the Hymenopteran homolog of the Drosophila centrosomal protein Dgrip84 localizes on the outer surfaces of AN. These organelles disintegrate in the late oocyte, leaving behind small gamma-tubulin foci, which likely seed the formation of maternal centrosomes. Accessory nuclei, therefore, may have played a significant role in the evolution of haplodiploidy in Hymenopteran insects (Ferree, 2006).

Gamma tubulin in vertebrates

continued: see Evolutionary Homologs part 2/2

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