gammaTubulin at 23C


Gamma tubulin in vertebrates

Gamma-Tubulin is a phylogenetically conserved component of microtubule-organizing centers that is essential for viability and microtubule function. Expression of a human gamma-tubulin cDNA restores viability and a near-normal growth rate to cells of S. pombe lacking endogenous gamma-tubulin. Immunofluorescence microscopy shows that these cells contain normal mitotic spindles and interphase microtubule arrays, and that human gamma-tubulin, like S. pombe gamma-tubulin, localizes to spindle pole bodies, the fungal microtubule-organizing centers. These results demonstrate that human gamma-tubulin functions in fission yeast, and suggest that in spite of the great morphological differences between the microtubule-organizing centers of humans and fission yeasts, gamma-tubulin is likely to perform the same tasks in both. They suggest, moreover, that the proteins that interact with gamma-tubulin must also be conserved, including, most obviously, microtubule-organizing center proteins. A fivefold overexpression of S. pombe gamma-tubulin causes no reduction in growth rates or alteration of microtubule organization. It is throught that excess gamma-tubulin is maintained in the cytoplasm in a form incapable of nucleating microtubule assembly (Horio, 1994)

Pericentrin and gamma-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. The relationship between these proteins was studied in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins are part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin-gamma-tubulin complex is distinct from the previously described gamma-tubulin ring complex (gamma-TuRC), because purified gamma-TuRC fractions do not contain detectable pericentrin. When assembled at the centrosome, the two proteins remain in close proximity, as shown by fluorescence resonance energy transfer. The three-dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis reveals a novel reticular lattice that is conserved from mammals to amphibians, and is organized independent of centrioles. The lattice changes dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appear to contact the minus ends of nucleated microtubules. These results indicate that pericentrin and gamma-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome (Dictenberg, 1998).

The localization of RNAs at the vegetal cortex in Xenopus oocytes is a complex process, involving at least two different pathways. The early, or messenger transport organizer (METRO), pathway localizes RNAs such as Xlsirts, Xcat2 and Xwnt11 during stages 1 and 2 of oogenesis, while the late pathway localizes RNAs such as Vg1 during stages 2-4. The onset of Vg1 localization is characterized by its microtubule-independent binding to a subdomain of the endoplasmic reticulum (ER). The formation of this unique ER structure is intimately associated with the movement of the mitochondrial cloud toward the vegetal cortex. In addition, the mitochondrial cloud contains a gamma-tubulin-positive structure that may function as a microtubule organizing center for establishing microtubule tracks for Vg1 localization. These data support, although they do not prove, a model in which the development of the late pathway machinery relies on the prior functioning of the early pathway (Kloc, 1998).

The in vivo function of gamma-tubulin in mammalian cells was investigated using a synthetic peptide to generate a polyclonal antibody that binds to a highly conserved segment of gamma-tubulin. After microinjection into cultured mammalian cells, immunofluorescence localization reveals that this antibody binds to native centrosomes at all phases of the cell cycle. In the presence of the gamma-tubulin antibody, microtubules fail to regrow into cytoplasmic arrays after depolymerization induced by nocodazole or cold. Furthermore, cells injected immediately before or during mitosis fail to assemble a functional spindle. Thus in vivo gamma-tubulin is required for microtubule nucleation throughout the mammalian cell cycle (Joshi, 1992).

Animal cells undergoing cytokinesis form the midbody, an inter-cellular bridge containing two bundles of microtubules interdigitated at their plus ends. Polyclonal antibodies raised against three specific amino acid sequences of gamma-tubulin specifically stained the centrosome in interphase, the spindle poles in all stages of mitosis, and the extremities of the midbody in mammalian cells. No gamma-tubulin could be detected in the interzone during anaphase and early telophase. Material containing gamma-tubulin first appears in the two daughter cells on each side of the division plane in late telophase, and for one hour after metaphase, accumulates transiently at the minus ends of the two microtubule bundles constituting the midbody. Micro-injection of gamma-tubulin antibodies into anaphase cells prevents the subsequent formation of the microtubule bundles between the two daughter cells. In contrast to previous views, these observations suggest that the microtubules constituting the midbody may be nucleated on special microtubule organizing centers, active during late telophase only, and assembled on each side of the dividing plane between the daughter cells (Julian, 1993).

To test whether gamma-tubulin mediates the nucleation of microtubule assembly in vivo, and co-assembles with alpha- and beta-tubulins into microtubules or self-assembles into macro-molecular structures, the expression of gamma-tubulin was elevated in the cell cytoplasm. In most cells, overexpression of gamma-tubulin causes a dramatic reorganization of the cellular microtubule network. When overexpressed, gamma-tubulin causes ectopic nucleation of microtubules that are not associated with the centrosome. In a fraction of cells, gamma-tubulin self-assembles into novel tubular structures with a diameter of approximately 50 nm (named gamma-tubules). Furthermore, unlike microtubules, gamma-tubules are resistant to cold or drug induced depolymerization. These data provide evidence that gamma-tubulin can cause nucleation of microtubule assembly and can self-assemble into novel tubular structures (Shu, 1995).

The highly conserved protein gamma-tubulin is required for microtubule nucleation in vivo. When viewed in the electron microscope, a highly purified gamma-tubulin complex from Xenopus consisting of at least seven different proteins is seen to have an open ring structure. This complex acts as an active microtubule-nucleating unit that can cap the minus ends of microtubules in vitro (Zheng, 1995).

Previous studies indicate that gamma tubulin ring complex (gammaTuRC) can nucleate microtubule assembly and may be important in centrosome formation. gammaTuRC contains approximately eight subunits, termed Xenopus gamma ring proteins (Xgrips), in addition to gamma tubulin. One gammaTuRC subunit, Xgrip109, is a highly conserved protein, with homologs present in yeast, rice, flies, zebrafish, mice, and humans. The yeast Xgrip109 homolog, Spc98, is a spindle-pole body component that interacts with gamma tubulin. In vertebrates, Xgrip109 identifies two families of related proteins. Xgrip109 and Spc98 have more homology to one family than the other. Xgrip109 is a centrosomal protein that directly interacts with gamma tubulin. A complementation assay for centrosome formation has been developed using demembranated Xenopus sperm and Xenopus egg extract. Using this assay, it has been shown that Xgrip109 is necessary for the reassembly of salt-disrupted gammaTuRC and for the recruitment of gamma tubulin to the centrosome. Xgrip109, therefore, is essential for the formation of a functional centrosome (Martin, 1998).

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. The 100- and 101-kD components of the gamma-tubulin complex have been identified as homologs of the yeast spindle pole body proteins Spc97p and Spc98p; the corresponding human proteins have been named hGCP2 and hGCP3. Sequence analysis reveals that these proteins are not only related to their respective homologs, 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 (Murphy, 1998).

A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method, using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies reveal that RanBPM is localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produces multiple spots that are colocalized with gamma-tubulin and act as ectopic microtubule nucleation sites, resulting in a reorganization of the microtubule network. RanBPM cosediments with the centrosomal fractions by sucrose-density gradient centrifugation. The formation of microtubule asters is inhibited not only by anti-RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacts with GTP-Ran in a two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin is faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM antibodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM (Nakamura, 1998).

Epithelial polarity, the ability of simple epithelial cells to become asymmetric, with distinct apical and basolateral domains in the plasma membrane, is a puzzling, as yet unsolved case of how cells can generate different subdomains. There is a consensus that epithelial cells sort out newly synthesized membrane proteins at the trans-Golgi network and recycle membrane proteins at an intermediate endosomal compartment. The vesicular traffic originating from these sorting compartments is probably directed by cytoskeletal components to their final destinations in the apical or basolateral domains and retained in place. Although epithelial cells can deliver polarized proteins without organized microtubules, several lines of evidence support the idea that microtubule based motors participate in the movement of exocytic vesicles, at least along part of their pathway. Moreover, in well-differentiated simple epithelia, microtubules are organized in the apico-basal axis with their minus ends toward the apical side. This organization is thought to participate in the polarization of organelles in the cytoplasm and to contribute to the polarized vesicular traffic. This peculiar arrangement of the microtubules must result from a polarized distribution of microtubule organizing centers (MTOC) under the apical domain. In fact, centrosomal structures distribute under the apical membrane in a number of simple epithelium cells in interphase, both ciliated and nonciliated. Interestingly, when epithelial cells enter mitosis, the centrosomes move toward the lateral domains, organizing the mitotic spindle always perpendicular to the apico-basal axis, and then move back to the apical domain when the cells complete mitosis. The significance of this elaborate redistribution of centrosomes is currently unknown. It is especially intriguing since apical (interphasic) centrosomes retain their capability to act as MTOC while most of the microtubules are organized by noncentrosomal MTOCs, diffusely distributed under the apical domain (Salas, 1999 and references).

A network of intermediate filaments (IF) underlying the apical membrane of epithelial cells contains cytokeratin (CK) 19 IF. These filaments are attached to a subpopulation of apical membrane proteins. Furthermore, this IF network, equivalent to the terminal web, is observed in a variety of epithelial cells with or without brush-border. Using antisense oligonucleotides, CK19 could be transiently downregulated in a fraction of CACO-2 epithelial cells. Those cells display a characteristic phenotype, with abolished apical F-actin (while the rest of cortical F-actin is normal), distinctive changes in the polarization of membrane proteins, and disorganization of the apical, but not basal, network of microtubules. The latter observation, together with the reports of an apical distribution of MTOC, prompted a hypothesis that the apical IF may be responsible for the polarized distribution of gamma-tubulin containing structures in simple epithelia. It is generally accepted that gamma-tubulin is an essential component of MTOC. It remains associated with the centrosomes even when the microtubules are depolymerized. Approximately 50% of the gamma-tubulin is in a soluble form, part of a 28S complex, while the rest is insoluble, presumably associated with centrosomes. Therefore, the possibility of MTOC binding to apical IF was analyzed, by studying the attachment of insoluble gamma-tubulin to CKs (Salas, 1999 and references).

The apical distribution of gamma-tubulin containing structures (potential microtubule-organizing centers) in CACO-2 cells was confirmed and perfect colocalization of centrosomes was demonstrated and nearly 50% of noncentrosomal gamma-tubulin with apical intermediate filaments, but not with apical F-actin. Furthermore, the antisense-oligonucleotide mediated downregulation of cytokeratin 19, using two different antisense sequences, is more efficient than anticytoskeletal agents to delocalize centrosomes. Electron microscopy colocalization suggests that binding occurs at the outer boundary of the pericentriolar material. Type I cytokeratins 18 and 19 present in these cells specifically coimmunoprecipitates in multi-protein fragments of the cytoskeleton with gamma-tubulin. The size and shape of the fragments, visualized at the EM level, indicate that physical trapping is an unlikely explanation for this result. Drastic changes in the extraction protocol do not affect coimmunoprecipitation. These results from three independent techniques indicate that insoluble gamma-tubulin containing structures are attached to apical intermediate filaments (Salas, 1999).

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. Two new human tubulins have been identified and they are shown to be are associated with the centrosome. One is a homolog of the Chlamydomonas delta-tubulin Uni3, and the other is a new tubulin, which has been 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 (Chang, 2000).

The first delta-tubulin described, Uni3 from Chlamydomonas, was identified from a mutant with defects in flagellar biogenesis and in the structure of the triplet microtubules of the basal body/centriole. The localization of human delta-tubulin to the region around the centrioles points to a potentially similar role for this protein in animal cells; however, the prominent intercentriolar localization of delta-tubulin suggests other possible functions as well. For example, in Clamydomonas, fibers linking the two centrioles within the centrosome have been found, and in vitro studies of centrosome duplication indicate that separation of the centrioles, and thus dissolution of the fibers, may be an early regulated step in the process. The unusual localization pattern of delta-tubulin resembles in several respects that observed for Skp1, a component of the SCF ubiquitin ligase, which is required for centriole separation (Chang, 2000).

epsilon-Tubulin has not been described previously: the only clue to its function comes from the cell-cycle-specific localization. Given that the single centrosome of G1 cells begins the cycle having epsilon-tubulin, the simplest interpretation of the differential localization in S and early G2 is that the pericentriolar material of one centrosome is different from that of the other after duplication. The newer centrosome, defined by the age of the parental centriole within the centrosome, acquires epsilon-tubulin only after some maturation process. This maturation is independent of the acquisition of nucleating capacity by the new centrosome, but might reflect some other function that varies with the cell cycle. The differential localization of epsilon-tubulin to old and new centrosomes is particularly interesting in light of the finding (Lange, 1995) that cenexin is a protein that associates only with the older centriole within a centrosome. Centriole distribution is semi-conservative; after centriole separation, the newer centriole of the original pair does not acquire cenexin until the G2/M transition, roughly at the same time that the new centrosome has acquired a full complement of epsilon-tubulin. The epsilon-tubulin results also indicate that duplication of the centrosome does not involve merely partitioning of the existing pericentriolar material around the two pairs of centrioles resulting from duplication, but rather that the centrosome containing the newer centriole also has 'new' pericentriolar material. The functional consequences of the difference between the duplicated centrosomes remain to be determined, but an attractive possibility is that it might be involved in the regulation of a cell-cycle-specific function, such as centrosome duplication, or cell-cycle-specific recruitment of other molecules to the centrosome (Chang, 2000).

This study shows that cytoplasmic dynein mediates assembly of pericentrin and gamma tubulin onto centrosomes. Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. In somatic cells, centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies it is concluded that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation (Young, 2000).

Based on this data, a model is proposed for the assembly of microtubule nucleating proteins. In this model, pericentrin binds to dynein through the light intermediate chain and to the gamma tubulin ring complex (gamma TuRC) through specific subunits of this complex. Dynein would mediate binding of the large pericentrin-gamma TuRC complex to microtubules and direct transport of the complex to centrosomes. At the centrosome, pericentrin-gamma TuRC complexes would be anchored, whereas dynein could be released for additional rounds of transport or anchored to perform additional roles. Dynactin may facilitate microtubule association or processivity of dynein and may contribute to centrosomal anchoring of gamma tubulin. This work raises the possibility that pericentrin mediates centrosome and spindle function through dynein-dependent assembly of microtubule nucleating complexes and other activities (Young, 2000).

There is now good evidence for microtubule-dependent and microtubule-independent mechanisms for recruitment of proteins onto centrosomes. These studies support the idea that dynein-mediated and passive diffusion mechanisms represent parallel pathways for centrosome assembly. It is possible that one pathway predominates over the other in certain biological systems or at different stages of the cell cycle. In embryonic systems, for example, high levels of centrosome proteins may be sufficient to drive the initial stages of microtubule-independent recruitment onto centrioles, although dynein-mediated transport becomes a major contributor at later times. Alternative mechanisms could also account for centrosome protein recruitment. Spontaneously assembled microtubules could be capped by gamma tubulin (and pericentrin) complexes, and these small microtubule fragments could be transported toward the minus ends of microtubules by dynein as described during spindle assembly in Xenopus extracts. These data do not distinguish between this microtubule fragment mechanism and the model presented in this paper in which presumably inactive centrosome proteins are transported to centrosomes and become active for microtubule nucleating activity. Another possibility is that centrosome-nucleated microtubules are released but remain tethered to the centrosome, perhaps through an interaction with dynactin, and they provide new minus ends for binding of gamma tubulin-pericentrin complexes after passive diffusion to these sites. Although this mechanism could account for the microtubule dependency of centrosome protein recruitment, it is inconsistent with kinetic data showing directed movement of GFP-pericentrin toward centrosomes (Young, 2000).

The topography of microtubule assembly events during meiotic maturation of animal oocytes demands tight spatial control and temporal precision. To better understand what regulates the timing and location of microtubule assembly, synchronously maturing mouse oocytes were evaluated with respect to gamma-tubulin, pericentrin, and total tubulin polymer fractions at specific stages of meiotic progression. gamma-Tubulin remains associated with cytoplasmic centrosomes through diakinesis of meiosis-1. Following chromatin condensation and perinuclear centrosome aggregation, gamma-tubulin relocates to a nuclear lamina-bounded compartment in which meiosis-1 spindle assembly occurs. gamma-Tubulin is stably associated with the meiotic spindle from prometaphase-1 through to anaphase-2, but also exhibits cell cycle-specific relocalization to cytoplasmic centrosomes. Specifically, anaphase onset of both meiosis-1 and -2 is characterized by the concomitant appearance of gamma-tubulin and microtubule nucleation in subcortical centrosomes. Brief pulses of taxol applied at specific cell cycle stages enhances detection of gamma-tubulin compartmentalization, consistent with a gamma-tubulin localization-dependent spatial restriction of microtubule assembly during meiotic progression. In addition, a taxol pulse during meiotic resumption impairs subsequent gamma-tubulin sorting, resulting in monopolar spindle formation and cell cycle arrest in meiosis-1; despite cell cycle arrest, polar body extrusion occurred roughly on schedule. Therefore, sorting of gamma-tubulin is involved in both the timing of location of meiotic spindle assembly as well as the coordination of karyokinesis and cytokinesis in mouse oocytes (Combelles, 2001).

Since gamma-tubulin is a known rate-limiting determinant of microtubule nucleation, it was postulated that redistribution of gamma-tubulin could underlie maturation-associated changes in microtubule patterning. The main findings of this study include: (1) redistribution of gamma-tubulin from predominantly cytoplasmic centrosomes to a nuclear compartment delimited by a persistent nuclear lamina; (2) sorting of gamma-tubulin between cytoplasmic and nuclear (spindle) compartments during meiotic progression based on the reappearance of gamma-tubulin in cytoplasmic centrosomes during anaphase of meiosis-1 and -2, and (3) functional uncoupling of karyokinesis and cytokinesis as a result of impaired sorting of gamma-tubulin after application of brief pulses of taxol. Together, these observations suggest that modifications in microtubule patterning during meiotic maturation are due to temporally and spatially regulated changes in gamma-tubulin distribution (Combelles, 2001).

Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), has been localized to the centrosome via the carboxyl-terminal region. This region interacts with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of kendrin, another centrosomal coiled-coil protein. The amino-terminal region of either CG-NAP or kendrin indirectly associates with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin coimmunoprecipitate with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins localize to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibits the microtubule nucleation; moreover, the combination of these antibodies results in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC (Takahashi, 2002).

Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. This study, carried out with Xenopus extracts, shows that the centrosomal coiled-coil protein pericentrin anchors gammaTuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupts gamma tubulin localization and spindle organization in mitosis but has no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupts the endogenous pericentrin-gammaTuRC interaction and perturbs astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncouples gammaTuRCs from centrosomes, inhibits microtubule aster assembly, and induces rapid disassembly of preassembled asters. All phenotypes are significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and are specific for mitotic centrosomal asters, since little effect was observed on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induces G2/antephase arrest followed by apoptosis in many but not all cell types. It is concluded that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death (Zimmerman, 2004).

A centrosome-independent role for γ-TuRC proteins in the spindle assembly checkpoint

The spindle assembly checkpoint guards the fidelity of chromosome segregation. It requires the close cooperation of cell cycle regulatory proteins and cytoskeletal elements to sense spindle integrity. The role of the centrosome, the organizing center of the microtubule cytoskeleton, in the spindle checkpoint is unclear. This study found that the molecular requirements for a functional spindle checkpoint included components of the large gamma-tubulin ring complex. However, their localization at the centrosome and centrosome integrity were not essential for this function. Thus, the spindle checkpoint can be activated at the level of microtubule nucleation (Muller, 2006).

Polo-like kinase 1 regulates protein that interact with gamma Tubulin

In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). A centrosomal Plk1 substrate has been identified, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. It is proposed that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity (Casenghi, 2003).

MT nucleation from the animal centrosome clearly depends on γ-TuRCs, but the mechanisms regulating the recruitment of these complexes to the centrosome remain poorly understood. A 156 kDa centrosomal protein, Nlp, has been identified whose properties suggest that it functions as a docking protein for γ-TuRCs during interphase of the cell cycle. Nlp displays significant structural similarity to ninein, a protein implicated in the capping and anchoring of MT minus ends at both centrosomal and noncentrosomal sites. Thus, the mammalian ninein family comprises at least two members, both of which appear to play important roles in the organization of MT arrays. The centrosome association of Nlp is regulated during the cell cycle. Nlp is a substrate of Plk1 and dissociates from centrosomes in response to phosphorylation, suggesting that Plk1 triggers an exchange of gamma tubulin binding proteins at the centrosome. Such an exchange of critical PCM components is likely to constitute a key aspect of centrosome maturation (Casenghi, 2003).

With Spc110p and Spc72p, two gamma tubulin binding proteins have been identified and characterized in S. cerevisiae, but the identification of gamma tubulin binding proteins in other organisms has proven difficult. Spc110 displays some sequence similarity with kendrin/pericentrin-B, but this similarity is largely restricted to a putative calmodulin binding domain. Additional mammalian proteins, including members of the pericentrin/kendrin/CG-NAP family, Cep135 and CPAP (centrosomal P4.1-associated protein), have been proposed to bind to γ-tubulin, but their precise contributions to MT organization remain to be clarified. The present study identifies Nlp (the product of cDNA KIAA0980) as a candidate gamma tubulin binding proteins in human cells. Nlp recruits both γ-tubulin and hGCP4, both in vitro and in vivo, suggesting that Nlp binds the entire γ-TuRC. Indeed, Nlp assemblies promoted MT nucleation both in mammalian cells and in Xenopus egg extracts. Conversely, microinjection of antibodies against Nlp severely suppressed MT nucleation (Casenghi, 2003).

Over its N-terminal half, Nlp shares 37% identity with ninein. However, ninein is substantially larger than Nlp, and the C termini of the two proteins show no structural homology, except for the presence of predicted coiled-coil domains. Ninein has been proposed to function in MT anchoring rather than MT nucleation. It is difficult to rigorously distinguish various MT minus end-associated activities and it would be premature to exclude that Nlp may also contribute to MT anchoring. Immunolocalization data suggest a preferential association of Nlp with one of the two centrioles, which might be consistent with an anchoring function. However, the data strongly indicate that Nlp plays an important role in the recruitment of γ-TuRCs to the centrosome. Thus, while it is clear that both Nlp and ninein play important roles in the organization of MT networks in mammalian cells, the two proteins may have functionally diverged during evolution (Casenghi, 2003).

Both yeast two-hybrid and direct biochemical data identify Nlp as a physiological substrate of Plk1. Furthermore, the results suggest that phosphorylation by Plk1 regulates the interaction of Nlp with both centrosomes and γ-TuRCs. In contrast, there is no evidence that Plk1 phosphorylates ninein, suggesting that Nlp and ninein are regulated differently. The analysis of Nlp mutants with in vitro Plk1 phosphorylation sites altered to alanine strongly suggests that Nlp is a direct substrate of Plk1 not only in vitro but also in vivo. These results also strengthen the view that the motif [E/DxS/T] constitutes a consensus for Plk1 phosphorylation sites. Although still tentative, the availability of such a consensus sequence may facilitate the future analysis of Plk1 substrates (Casenghi, 2003).

The abrupt increase in the MT nucleation activity of centrosomes at the onset of mitosis is expected to require substantial changes in pericentriolar material composition. These are apparently controlled by several protein kinases, including Plk1, Aurora-A, and Nek2, but only a few substrates of these kinases have so far been identified. Nlp properties suggest that it functions in MT nucleation at the centrosome. Remarkably, however, Nlp is displaced from the centrosome at the onset of mitosis, when centrosomal MT nucleation activity increases dramatically. It follows that Nlp functions in centrosomal MT nucleation specifically during interphase (G1, S, and G2) of the cell cycle, but not during M phase. This implies that structurally distinct gamma tubulin binding proteins function as interphasic and mitotic scaffolds for γ-TuRC recruitment. These data further suggest that the activation of Plk1 at the G2/M transition results in the displacement of Nlp from the maturing centrosome and that this event is important for mitotic spindle formation. It is envisioned that the removal of Nlp from the centrosome constitutes a prerequisite for the recruitment of an as yet unidentified mitotic gamma tubulin binding proteins, which then confers enhanced microtubule nucleation capacity to the centrosome. According to this model, the activation of Plk1 at the onset of mitosis triggers the replacement of the interphasic MT nucleation scaffold by the mitotic scaffold (Casenghi, 2003).

γ-Tubulin plays an essential role in the coordination of mitotic events

Recent data from multiple organisms indicate that gamma-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, the phenotype of mipAD159, a cold-sensitive allele of the gamma-tubulin gene of Aspergillus nidulans, was examined. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (approximately 6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that gamma-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control (Prigozhina, 2004).

Microtubule nucleation and anchoring at the centrosome are independent processes linked by ninein function

The centrosome organizes microtubules by controlling nucleation and anchoring processes. In mammalian cells, subdistal appendages of the mother centriole are major microtubule-anchoring structures of the centrosome. It is not known how newly nucleated microtubules are anchored to these appendages. Ninein, a component of subdistal appendages, localizes to the centriole via its C-terminus and interacts with gamma-tubulin-containing complexes via its N-terminus. Expression of a construct encoding the ninein C-terminus displaces endogenous ninein and the gamma-tubulin ring complex (gamma-TuRC) from the centrosome, leading to microtubule nucleation and anchoring defects. By contrast, expression of a fusion consisting of the N- and C-terminal domains (lacking the central coiled-coil region) displaces endogenous ninein without perturbing gamma-TuRC localization. Accordingly, only anchoring defects are observed in this case. Therefore, expression of this fusion appears to uncouple microtubule nucleation and anchorage activities at the centrosome. These results suggest that ninein has a role not only in microtubule anchoring but also in promoting microtubule nucleation by docking the gamma-TuRC at the centrosome. In addition, the gamma-TuRC might not be sufficient to anchor microtubules at the centrosome in the absence of ninein. It is therefore propose that ninein constitutes a molecular link between microtubule-nucleation and -anchoring activities at the centrosome (Delgehyr, 2005).

The number of microtubules nucleated at the centrosome is probably much higher than the number of anchoring sites available at the tips of the nine appendages. One must therefore postulate a selective stabilization of microtubules at anchoring sites rather than a simplistic pathway of nucleation followed by anchoring. Interestingly, a proportion of the centrosomal gamma-tubulin has been observed at the tips of subdistal appendages. Moreover, proteins already implicated in microtubule anchoring such as p150Glued or EB1, which are localized to the mother centriole, also have microtubule-nucleating properties. These data suggest that a subset of microtubules could be nucleated and later anchored at the subdistal appendages. Based on the current results, it is proposed that microtubules nucleated in a gamma-TuRC-dependent manner at the centrosome would have different behaviours. Microtubules nucleated far from the subdistal appendages would be transiently anchored by the gamma-TuRC but then released from the centrosome in a manner possibly dependent on specific factors. Indeed, many microtubule seeds are produced and released from the centrosome. By contrast, microtubules nucleated at the vicinity or on the subdistal appendages would become anchored more readily at these structures on the mother centriole. Therefore, ninein, a component of these appendages, might favour microtubule nucleation (by docking the gamma-TuRC), followed by anchoring. These newly formed microtubules might then be anchored directly, owing to ninein function, or might require additional elements for the transfer to anchorage sites. Interestingly, in ear pillar cells, microtubules are anchored at a considerable distance from the centrosome and it is possible that similar modes of microtubule activate transfer occur at the centrosome (Delgehyr, 2005).

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

The γ-tubulin ring complex (γTuRC) is a large multi-protein complex that is required for microtubule nucleation from the centrosome. The GCP-WD protein (originally named NEDD1) is the orthologue of the Drosophila Dgrip71WD protein, and is a subunit of the human γTuRC. GCP-WD has the properties of an attachment factor for the γTuRC: depletion or inhibition of GCP-WD results in loss of the γTuRC from the centrosome, abolishing centrosomal microtubule nucleation, although the γTuRC 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 γ-tubulin with the spindle, separately from association with the centrosome. These results indicate that GCP-WD broadly mediates targeting of the γTuRC to sites of microtubule nucleation and to the mitotic spindle, which is essential for spindle formation (Luders, 2006).

Centrosomal microtubule nucleation is mediated by a large protein complex, termed the γ-tubulin ring complex (γTuRC). The γTuRC consists of the tubulin superfamily member γ-tubulin, and additional subunits, named gamma complex proteins (GCPs) 2-6 in human cells. The known GCPs are related to each other, and represent a conserved protein family that are found in all eukaryotes, although the total number of such proteins varies (Luders, 2006).

In many cell types, nucleation takes place at sites other than the centrosome: for example, at the surface of the nuclear envelope in vertebrate myotubes and in plant cells. In all characterized cases, γ-tubulin is present at these non-centrosomal sites of nucleation, but the mechanism of targeting to any site is not known (Luders, 2006).

Most of the γTuRCs in interphase cells are cytoplasmic, but there is a rapid 3-5-fold increase in the amount of centrosomal γ-tubulin at the onset of mitosis, which is consistent with cell-cycle-regulated recruitment of the γTuRC. γTuRC also associates with the mitotic spindle. Whether this simply reflects capping of spindle microtubule minus ends by the γ-TuRC or whether it involves specific spindle interactions remains unclear. These changes in γTuRC localization in mitosis are probably regulated by mitotic kinases, but relevant substrates have not been identified (Luders, 2006).

Recently, a new component of the Drosophila γTuRC, Dgp71WD, was identified (Gunawardane, 2003). This study characterizes the human protein GCP-WD and shows that it is the orthologue of Dgp71WD. GCP-WD is required for microtubule nucleation both at centrosomes and at mitotic chromatin in human cells. Spindle localization of the γTuRC is regulated by mitotic phosphorylation of GCP-WD. These data indicate a new function for the γTuRC in spindle formation (Luders, 2006).

Human GCP-WD, the orthologue of Drosophila Dgp71WD, is a subunit of the human γTuRC that is required for its localization to the centrosome and to the mitotic spindle. It is proposed that GCP-WD is a targeting factor for the γTuRC, which is involved in the spatial and temporal control of microtubule nucleation (Luders, 2006).

Interphase centrosomes do not form microtubule asters when GCP-WD is depleted or inhibited; however, the assembly of cytoplasmic microtubules is not affected. A formal possibility is that the cytoplasmic microtubules are derived from centrosomal nucleation and release. This is considered unlikely for two reasons: (1) at 10 s of microtubule regrowth, there are centrosomal microtubules in control cells, but no cytoplasmic microtubules in GCP-WD-depleted cells; and (2) as soon as cytoplasmic microtubules appear (at the 30 s time point), they are distributed throughout the cell; this is inconsistent with the reported rate of movement of released microtubules. The data indicate the existence of centrosome-independent mechanisms of microtubule nucleation, in agreement with results from γ-tubulin depletion and centrosome-ablation experiments (Luders, 2006).

Depletion of centrosomal γ-tubulin by RNAi does not affect the centrosomal localization of GCP-WD; this is consistent with the proposed centrosomal attachment function. Several centrosome proteins have been proposed to recruit the γTuRC to the centrosome and, therefore, are potential binding partners of GCP-WD, although alternative means of attaching the γTuRC to centrosomes may exist. In addition, it will be important to determine the role of GCP-WD in γTuRC targeting in cell types in which microtubule nucleation occurs from sites other than the centrosome (Luders, 2006).

Mitotic chromatin-mediated microtubule nucleation during microtubule regrowth has been demonstrated previously and depletion of GCP-WD has revealed a role in this process. Chromatin-mediated nucleation, which is controlled by activated Ran GTPase, requires Ran-dependent spindle-assembly factors such as TPX2, and also seems to involve γ-tubulin. The requirement for GCP-WD in this process indicates that specific targeting of the γTuRC is necessary, rather than its mere presence in the cytoplasm (Luders, 2006).

In addition to centrosomal localization, γTuRC components have been shown to localize to the mitotic spindle, seeming to overlap with kinetochore microtubules. Two scenarios can be envisioned for the roles of non-centrosomal GCP-WD and γTuRC in spindle formation. In the first scenario, the γTuRC simply reports the positions of microtubule minus ends in the spindle. These microtubules might have been nucleated from the centrosome or the chromatin, released and incorporated into the spindle. This model is consistent with the observation that katanin inhibition results in decreased spindle γ-tubulin, and with the number and distribution of minus ends in the spindle of Ptk1 cells, most of which are located within the kinetochore microtubule bundles. Phosphorylation of GCP-WD at Ser 418 might be required for some aspect of this microtubule-end bundling, without which a robust spindle cannot form. In this model, detection of γTuRC components in the spindle depends on the presence of bundled minus ends. In the second scenario, the observed distribution of γTuRC in the spindle reflects lateral association with microtubules, as has been reported for γ-tubulin. In this model, phosphorylation of GCP-WD is required for the lateral interaction of the γTuRC with microtubules either by direct binding, or indirectly through associated proteins. A possible function for this mode of binding would be the nucleation of additional microtubules by the γTuRC along pre-existing microtubules, as observed in Schizosaccharomyces pombe. Such an amplification mechanism for chromatin-mediated microtubule nucleation might increase the efficiency of microtubule bundling. The two scenarios for GCP-WD and γTuRC function in the spindle are not mutually exclusive and could both contribute to the observed properties (Luders, 2006).

Although data supporting a role for phosphorylation in the recruitment of γ-tubulin to mitotic centrosomes have been reported previously, the GCP-WD S418A mutation only affects association of GCP-WD and γ-tubulin with the mitotic spindle, and not the centrosome. Future studies will reveal whether GCP-WD plays a role in γTuRC targeting not only in the cell cycle, but also in developmental processes, possibly involving additional sites of phosphorylation or modification (Luders, 2006).

Shroom family proteins regulate gamma-tubulin distribution and microtubule architecture during epithelial cell shape change

Cell shape changes require the coordination of actin and microtubule cytoskeletons. The molecular mechanisms by which such coordination is achieved remain obscure, particularly in the context of epithelial cells within developing vertebrate embryos. A role has been identifed for the novel actin-binding protein Shroom3 as a regulator of the microtubule cytoskeleton during epithelial morphogenesis. Shroom3 is sufficient and also necessary to induce a redistribution of the microtubule regulator gamma-tubulin. Moreover, this change in gamma-tubulin distribution underlies the assembly of aligned arrays of microtubules that drive apicobasal cell elongation. Finally, experiments with the related protein, Shroom1, demonstrate that gamma-tubulin regulation is a conserved feature of this protein family. Together, the data demonstrate that Shroom family proteins govern epithelial cell behaviors by coordinating the assembly of both microtubule and actin cytoskeletons (Lee, 2007).

Plk4-induced centriole biogenesis in human cells

Overexpression of Polo-like kinase 4 (Plk4) in human cells induces centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole. This provided an opportunity for dissecting centriole assembly and characterizing assembly intermediates. Critical components were identified and ordered into an assembly pathway through siRNA and localized through immunoelectron microscopy. Plk4, hSas-6, CPAP, Cep135, gamma-tubulin, and CP110 (see Drosophila CP110) were required at different stages of procentriole formation and in association with different centriolar structures. Remarkably, hSas-6 associated only transiently with nascent procentrioles, whereas Cep135 and CPAP formed a core structure within the proximal lumen of both parental and nascent centrioles. Finally, CP110 is recruited early and then associates with the growing distal tips, indicating that centrioles elongate through insertion of α-/β-tubulin underneath a CP110 cap. Collectively, these data afford a comprehensive view of the assembly pathway underlying centriole biogenesis in human cells (Kleylein-Sohn, 2007).

Centriole biogenesis in mammalian cells remains poorly understood, but substantial progress has recently been made in invertebrate organisms. In Caenorhabditis elegans, a protein kinase, Zyg-1, and four putative structural proteins, termed SPD-2, Sas-4, Sas-5, and Sas-6, are required for centriole duplication. Moreover, through elegant epistasis experiments and electron tomography the five proteins could be shown to assemble sequentially on nascent procentrioles. Independently, the protein kinase Plk4 (also known as Sak) has been identified as a key regulator of centriole duplication in both Drosophila and human cells. Although the two kinases lack obvious sequence homology, it is plausible that Plk4 represents a functional homolog of C. elegans Zyg-1. When overexpressed in unfertilized eggs of Drosophila, Plk4 (Sak) induces the de novo formation of centrioles, demonstrating that this kinase is able to induce centriole biogenesis even in the absence of pre-existing centrioles. Homologs of nematode Sas-4 and Sas-6 were also required for centriole biogenesis in Drosophila (see Drosophila Sas-4), and a requirement for Sas-6 was demonstrated for human cells, suggesting that fundamental aspects of centriole biogenesis have most likely been conserved during evolution (Kleylein-Sohn, 2007).

Overexpression of Plk4 in human cells causes the recruitment of electron-dense material onto the proximal walls of parental centrioles, suggesting that Plk4 is able to trigger procentriole formation. This study used a cell line allowing the temporally controlled expression of Plk4 to study the formation of centrioles in human cells. Plk4 is shown to trigger the simultaneous formation of multiple procentrioles around each pre-existing centriole. These multiple centrioles form during S phase and persist as flower-like structures throughout G2 and M phase before they disperse in response to disengagement during mitotic exit, giving rise to a typical centriole amplification phenotype. Through siRNA-mediated depletion of individual centrosomal proteins, several gene products important for Plk4-controlled centriole biogenesis have been identified and assigned individual proteins to distinct steps in the assembly pathway. Finally, these functional data have been correlated with morphological analyses using immunoelectron microscopy. Taken together, these results provide a first molecular analysis of centriole formation in human cells (Kleylein-Sohn, 2007).

Rab11 endosomes contribute to mitotic spindle organization and orientation

During interphase, Rab11-GTPase-containing endosomes recycle endocytic cargo. However, little is known about Rab11 endosomes in mitosis. This paper show that Rab11 localizes to the mitotic spindle and regulates dynein-dependent endosome localization at poles. Mitotic recycling endosomes were found to bind gamma-TuRC components and associate with tubulin in vitro. Rab11 depletion or dominant-negative Rab11 expression disrupts astral microtubules, delays mitosis, and redistributes spindle pole proteins. Reciprocally, constitutively active Rab11 increases astral microtubules, restores gamma-tubulin spindle pole localization, and generates robust spindles. This suggests a role for Rab11 activity in spindle pole maturation during mitosis. Rab11 depletion causes misorientation of the mitotic spindle and the plane of cell division. These findings suggest a molecular mechanism for the organization of astral microtubules and the mitotic spindle through Rab11-dependent control of spindle pole assembly and function. It is proposes that Rab11 and its associated endosomes cocontribute to these processes through retrograde transport to poles by dynein (Hehnly, 2014).

back to Evolutionary Homologs part 1/2

gammaTubulin at 23C: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.