BIOLOGICAL OVERVIEW In eukaryotic cells, a specialized organelle called the microtubule organizing center (MTOC) is responsible for the disposition of microtubules, helping to form them into a radial, polarized array in interphase cells and in the spindle of mitotic cells. Eukaryotic cells across different species, and different cell types within single species, have morphologically diverse MTOCs, but all these MTOCs share in common the function of organizing microtubule arrays. MTOCs effect microtubule organization by initiating microtubule assembly and anchoring microtubules at their slowly growing minus ends, thus ensuring that in each microtubule array it is the rapidly growing plus ends that extend distally. Gamma-Tubulin is found in the MTOCs of cells from many different organisms, and has several properties that make it a good candidate for both initiation of microtubule assembly and anchorage (Joshi, 1993).

GammaTubulin was first identified as a suppressor of a ß-tubulin mutation in the fungus Aspergillus nidulins. Unexpectedly, antibodies raised against the protein do not stain microtubules, but instead stain the spindle pole body (the MTOC, or centrosome). In Drosophila, gammaTubulin forms a complex with two centrosomal microtubule-associated proteins called CP190 and CP60. Since CP60 can associate with microtubules, and the gammaTubulin-CP190-CP60 complex associates with microtubules, it is believed that the CP60 component binds the complex to microtubules. These observations suggest that gammaTubulin, CP190, and CP60 are all components of a centrosomal complex that can interact with microtubules (Raff, 1993).

CP60 is not homologous to any protein in the database, although it contains six consensus sites for phosphorylation by cyclin-dependent kinases. CP60 is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is maximal during anaphase and telophase, and then drops dramatically between late telophase and early interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase. CP60 isolated from Drosophila embryos is highly phosphorylated; dephosphorylated CP60 forms a good substrate for cyclin B/p34cdc2 kinase complexes. Another kinase activity capable of phosphorylating CP60 is present in the CP60/CP190 multiprotein complex. CP60 binds to purified microtubules; this binding is blocked by CP60 phosphorylation (Kellogg, 1995).

What is the function of gammaTubulin? The microtubule cytoskeleton in animal cells does not assemble spontaneously, but instead requires the function of a centrosome. This organelle consists of a pair of centrioles surrounded by a complex collection of proteins known as the pericentriolar material (PCM). The PCM is required for microtubule nucleation. During interphase, the minus (or slow-growing) ends of microtubules are embedded in the PCM and the plus (or fast-growing) ends project outwards into the cytoplasm (or during mitosis, into the spindle apparatus). gammaTubulin is the only component of the PCM thus far implicated in microtubule nucleation. GammaTubulin is localized in the PCM ring structures of purified Drosophila centrosomes. When these centrosomes are used to nucleate microtubule growth, gammaTubulin is localized at the minus ends of the microtubules (Moritz, 1995b).

The gammaTubulin complex has been purified from Xenopus egg extracts. The purified complex is a ring structure with a 25 to 28 nm diameter. Many of these rings appear to be open, with the ends overlapping each other. The wall of a ring is roughly cylindrical: and it is both wider than the wall of a microtubule and higher than an alpha/beta tubulin dimer. The complex nucleates microtubules in vitro, binding to one end of a microtubule. It is concluded that microtubule-nucleating sites within the PCM are ring-shaped templates containing multiple copies of gamma-tubulin. The outer diameter of the complex is similar to the outer diameter of a microtubule. Each complex also has less than two but more than one helical turn, allowing a fit to the end of a microtubule. It is thought that the CP60 and CP190 proteins described above define the framework of a helical structure to which gamma-tubulins bind. The gamma-tubulins then provide a seed for the assembly of alpha/beta tubulin dimers (See Drosophila beta1 Tubulin) that have been stabilized by their interactions with gamma-tubulin. It is thought that the gamma-tubulin complex constitutes the long sought after microtubule nucleating factor in the pericentriolar matrix (Zheng, 1995).

GammaTuRC is required to maintain juxtaposed half spindles in spermatocytes

The weak spindle integrity checkpoint in Drosophila spermatocytes has revealed a novel function of the gamma-tubulin ring complex (gammaTuRC) in maintaining spindle bipolarity throughout meiosis. Bipolar and bi-astral spindles form in Drosophila mutants for dd4, the gene encoding the 91 kDa subunit of gammaTuRC. However, these spindles collapse around metaphase and begin to elongate as if attempting anaphase B. The microtubules of the collapsing spindle fold back on themselves, their putative plus ends forming the focused apices of biconical figures. Cells with such spindles are unable to undergo cytokinesis. A second type of spindle, monopolar hemi-spindles, also forms as a result of either spindle collapse at an earlier stage or failure of centrosome separation. Multiple centrosome-like bodies at the foci of hemi-spindles nucleate robust asters of microtubules in the absence of detectable gamma-tubulin. Time-lapse imaging revealed these to be intermediates that develop into cones, structures that also have putative plus ends of microtubules focused at their tips. Unlike biconical figures, however, cones seem to contain a central spindle-like structure at their apices and undergo cytokinesis. It is concluded that spermatocytes do not need astral microtubules nucleated by opposite poles to intersect in order to form a central spindle and a cleavage furrow (Barbosa, 2003).

Weak hypomorphic alleles of dd4 have allowed a study of the stages of spermatogenesis that are most sensitive to the compromised function of the 91 kDa component of the gammaTuRC encoded by this gene. These mutant alleles appear usually able to provide sufficient functional protein for the four rounds of mitosis that precede meiosis but then show a variety of spindle defects during meiosis. This correlates with a loss of gamma-tubulin staining from the spindle pole, suggesting substantial disruption of the gammaTuRC. The spindle abnormalities displayed in meiosis contrast in several respects to those described in mitotic divisions. dd4 mutant larval neuroblasts arrest in mitosis at metaphase with bipolar spindles that have disorganized poles lacking gamma-tubulin and which do not have astral microtubules. dd4^S meiocytes also lack gamma-tubulin at their poles but, nevertheless, are capable of organizing arrays of astral microtubules. In contrast to dd4 mitotic cells, stable biastral structures either fail to form or they collapse after their formation. The results of combined application of real-time imaging of spermatocytes and immunolocalization of specific antigens in fixed preparations led to a model for how the various abnormalities of the meiotic spindle arise. In cells in which bi-astral spindles either never form or collapse very early, monopolar spindles first develop that are postulated to correspond to the hemi-spindles seen in fixed preparations. After some time it seems that these can develop bipolarity as a result of chromatin accumulating on their periphery and a rudimentary spindle midzone can form in such structures. These are one type of cone-like spindle that, in some cases, may even complete cytokinesis to generate aneuploid daughter cells. Such structures correspond to conical spindles described in testes from mutants of the gamma-tubulin gene at 23C (gammaTub23C^PI). The present work thus confirms that such structures arise from disruption of the gamma-TuRC and extends it by showing hemispindles to be an intermediate in the formation of cones. It also allows the demonstration of an alternative pathway by which conical structures can arise and thereby casts light on a novel role for the gamma-TuRC in maintaining the stability of co-joined hemi-spindle structures in the normal bipolar meiotic spindle. This is indicated by observations that bipolar spindles with well-defined poles could be formed but then collapse around the time of the metaphase-anaphase transition, causing the two poles to move back together. As a consequence, the intervening spindle microtubules are displaced and the central region of the spindle folds back on itself at two points to form the apices of biconical figures. Examination of the dd4 mutant phenotype in testes has thus permitted three types of spindle defects to be identified: within asters themselves; in the spindle microtubules required for centrosome separation, and in the central region of the spindle, each of which will be discussed (Barbosa, 2003).

The spindle poles of dd4 primary spermatocytes usually have the expected number of centrioles by the criteria of discrete bodies of centrosomin (CNN), a component of the pericentriolar material (PCM) that closely surrounds the centrioles in such cells. However, the finding of some spermatocytes with more than four such bodies suggests that there can be failure in centriole separation in the pre-meiotic divisions as has been described in mutant dd4 neuroblast divisions. The CNN-containing bodies in dd4 spermatocytes appear either to have never fully separated or have become reunited after spindle collapse and so the four such bodies are usually at the focus of the astral poles. In common with dd4 mutant neuroblasts, these pole bodies lack the gammaTuRC but are associated with Abnormal spindle (Asp). The ability of these poles to nucleate asters thus goes against the accepted dogma that the proper localization of gamma-tubulin and centrosomal integrity is absolutely required for the function of a polar MTOC to direct the formation of asters. At present it can only be speculated why astral microtubule arrays are not seen in dd4 neuroblasts and yet appear robust in spermatocytes mutant for the hypomorphic allele dd4^S. This could reflect a general deterioration of the spindle throughout a prolonged period of metaphase delay due to the more robust spindle integrity checkpoint in neuroblasts. However, it could also reflect underlying differences in spindle structure and function between these cell types. It is possible, for example, that Asp in the focus of asters in dd4^S spindles may play more of a role in maintaining astral microtubules in spermatocytes than it does in neuroblasts. This would be consistent with the known function of Asp in the reorganization of radial arrays of microtubules around isolated Drosophila centrosomes. Moreover, meiotic spindles in asp spermatocytes are abnormal in shape, and the morphology of their asters is considerably affected However, it would seem that Asp may not be as efficient at stabilizing asters in the dd4 larval CNS as in dd4 spermatocytes (Barbosa, 2003).

Many of the astral structures revealed by the immunostaining of dd4 testes appeared sufficiently asymmetric to have the appearance of hemi-spindles. These were truly monopolar by the criteria of having Asp at the focused putative minus ends of microtubules and with Pav-KLP located at their periphery, the putative plus ends. Such hemi-spindles are quite different structures from the asymmetric spindles sometimes observed in dd4 mutant neuroblasts in which one Asp containing pole can be focused and the other comprised of scattered bundles of microtubules whose putative minus ends are associated with Asp. However, real-time imaging suggests the hemi-spindles seen in dd4 meiocytes are an intermediary in the development of cones. In this process it seems that bipolarity is developed by the chromatin apparently acting to stabilize the diverging microtubules. Such spindles have one pole with multiple centrioles and the other with none (Barbosa, 2003).

The difficulties in either establishing or maintaining the separation of spindle poles in male meiosis in dd4 mutants point toward a novel role for the gammaTuRC in maintaining the function of spindle microtubules per se. It is possible that there could be two stages to this process that differ in their sensitivity to the compromised function of the gammaTuRC. This is suggested by the finding that in some cells, bipolar spindles either never form or collapse early (to form initially a hemi-spindle). Thus, the first crucial requirement of gamma-tubulin function may be to nucleate a subset of spindle microtubules that maintain bipolarity. If a bipolar and bi-astral spindle does form then it seems to undergo a crisis around metaphase when it appears to collapse. The collapsing spindles do elongate however, suggesting that collapse may in part be driven by anaphase events. In some ways the spindle collapse is reminiscent of the consequences of inactivating gamma-Tub function by RNAi in Caenorhabditis elegans embryos that result in separated asters re-approaching each other at late prophase. Moreover, conical spindles in gammaTub23C^PI spermatocytes seem to appear from a collapse of bipolar spindles around prophase and elongate in a timeframe comparable to the assembly of the central spindle in wild type. It is possible that a second, stabilizing effect of the gammaTuRC at the minus ends of the microtubules is specially required before metaphase in meiosis I. In vertebrate cells, low doses of taxol have been shown to preferentially stabilize kinetochore microtubules plus ends leading to a slight collapse of the spindle around the time of metaphase. Perhaps the reduction of centrosomal gammaTuRCs in gammaTub23C^PI and dd4 cells is reproducing this effect by destabilizing the minus ends of microtubules (Barbosa, 2003).

The normal origin of the central spindle microtubules in wild-type cells is obscure. Treatment of cells with microtubule destabilizing agents after the onset of anaphase suggests that the central spindle may be assembled from newly nucleated microtubules and not from remains of the mitotic spindle material left in the cell equator. However, although the localization of gamma-tubulin in the central spindle of mammalian dividing cells has been reported by several groups, the presence of gamma-tubulin in Drosophila central spindle is still a matter of debate. The spindle collapse that occurs in dd4 meiocytes could be related to the onset of reorganization of the spindle that occurs at the metaphase-anaphase transition when some microtubules appear to detach from the centrosomes as the central spindle structure begins to form. In wild-type meiocytes this is seen by the generation of a new set of central spindle microtubules with Asp at their putative minus ends. Central spindle microtubules never become fully organized in the dd4 spermatocytes although this seems to progress further in cones. Consistently, Asp never undertakes its normal redistribution but rather adopts a fibrous pattern of organization extending from the spindle poles. If as it has been suggested, Asp works as an anchor to the putative minus ends of microtubules, it is possible that microtubules are released from the spindle poles and rather dispersed throughout the conical microtubule structure in dd4 meiocytes. But the lack of Asp capped microtubules of central spindle-like structures in these cells suggests some degree of co-operation with the gamma-TuRC is necessary to correctly co-ordinate this transition in spindle structure (Barbosa, 2003).

Despite the absence of clearly organized central spindle microtubules, the mutant cells do show several features typical of post-metaphase stages of meiosis that differ in two pathways of spindle development. The hemi-spindles that give rise to cones harbor homologs that are initially mono-oriented as they move toward and away from the asters without evidence of segregation. As cones develop from the hemi-spindles, bipolarity appears to arise from some ability of chromosomes to stabilize microtubules as discussed above. At this time, microtubules stabilized by distal chromatin in some hemi-spindles would appear to interdigitate with microtubules from the astral pole in an anti-parallel manner to form cones with the motor protein Pav-KLP then becoming associated with a 'knot-like' structure at the center of the spindle but never forming a ring. Rings of septin and actin can then form around structures equivalent to those where the Pav-KLP 'knots' appear. Sometimes these enable cytokinesis to be achieved. In the pathway in which bipolar spindles collapse there is an elongation of spindle microtubules analogous to the lengthening that takes place in anaphase B. Such spindles have no arrangement of microtubules that resembles a central spindle. They lack the bipolarity usually associated with central spindle formation and unlike the hemi-spindles they appear to lack the ability for regenerating such a bipolar structure. The presence of Pav-KLP at the apices of the biconical figures suggests that although Pav-KLP is a known prerequisite for central spindle formation, this localization is in itself insufficient for this process. Thus, central spindle-like structures do not form in the biconical figures possibly reflecting the absence of interdigitating microtubules inherent in a bipolar structure and this in turn leads to a failure in formation of rings of septin and actin. Thus as observed in gammaTub23C^PI spermatocytes there seems to be some limited ability to organize some of the components required for cytokinesis when gammaTuRC function is compromised, the extent of which appears to reflect the ability to reorganize central spindle microtubules (Barbosa, 2003).

In summary these observations indicate that the gammaTuRC may provide several functions to the spindle. It is not absolutely essential for microtubule nucleation to form asters in all cell types. Rather, it may be required for the specific function of subsets of spindle microtubules that maintain pole separation. It appears to co-operate with other proteins associated with the minus ends of microtubules, notably Asp in Drosophila cells, and this appears to be important in the reorganization of the spindle that occurs following the metaphase-anaphase transition. Further work will be required to determine the extent to which defects in the reorganization of the central spindle at this stage reflect a direct requirement for the gammaTuRC or are a consequence of earlier defects in spindle organization (Barbosa, 2003).

Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics

The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. This study reports a novel role for the multifunctional adaptor protein centrosomes and promotes correct orientation of the MS in asymmetrically dividing Drosophila stem cells and epithelial cells, and symmetrically dividing Drosophila and human epithelial cells. ALIX-deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Drosophila Spindle defective 2 (DSpd-2)/Cep192, where ALIX promotes accumulation of gamma-tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting microtubule-associated protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, these results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division (Malerod, 2018).

During cell division, the mitotic spindle (MS) that forms between the two centrosomes ensures faithful segregation of the chromosomes between the two daughter cells, positions the cleavage furrow, and is anchored to the cell cortex to ensure proper spindle orientation. Different subpopulations of microtubules (MTs); the kinetochore, interpolar/astral, and astral MTs, are involved in controlling each process, respectively. Correct orientation of the MS ensures proper segregation of molecules defining cell fate and is important during asymmetric stem cell division to generate one daughter cell which self-renews and one which undergoes differentiation. The orientation of the MS further defines the cleavage plane of the cell and thereby its position within the tissue, exemplified by the planar division of epithelial cells to generate a monolayered epithelium. The precise orientation of the MS can be influenced by internal cues (cell polarity determinants) or external cues (neighboring cells or extracellular matrix) and is cell type-dependent (Malerod, 2018).

Regardless of the molecular mechanisms setting the orientation, the MS is anchored to the cell cortex by the astral MTs radiating from the centrosomes. The centrosome is the major MT-organizing center in most cell types and nucleates astral MTs and the other MT subpopulations of the MS. The centrosome is composed of a centriole pair and the surrounding pericentriolar material (PCM), generated by dynamic assembly of proteins found to stabilize each other via positive feedback loops. During mitosis, the centrosome matures when the PCM expands extensively due to recruitment of scaffold and MT nucleating proteins, which promote MS formation. The γ-tubulin ring complexes (γTuRCs) of the PCM, composed of γ-tubulin and associated proteins (γ-tubulin complex proteins, GCPs), nucleate MT filaments at the centrosome. The ring of γ-tubulin within γTuRC resembles the MT geometry and serves as a template for assembly of α/β-tubulin-dimers, which polymerize into long filaments, MTs. Although the centrosomes represent the major centers for MT nucleation, MTs may alternatively be formed at the Golgi, chromosomes, nuclear envelope, plasma membrane, and pre-existing MTs. Importantly, γ-tubulin seems to be implicated in the nucleation process regardless of the intracellular localization (Malerod, 2018).

Microtubules of the MS, including the astral MTs, are dynamic and their timely assembly and disassembly is tightly controlled by proteins regulating nucleation, severing, and stability of the filaments. MT stability is regulated by MT-associated proteins. These proteins stabilize MTs by binding to the growing plus-end of the filaments to prevent catastrophe, or alternatively, by decorating the MTs to prevent interaction with severing proteins. Furthermore, the γTuRC itself has also been reported to modulate the stability of MTs by interacting with motor proteins such as dynein, kinesin-5, and kinesin-14 as well as the plus-end tracking protein EB1 (Malerod, 2018).

Astral MT regulation occurs at several levels to achieve proper MS orientation: (1) astral MT nucleation at the centrosomes, (2) astral MT dynamics and stability, and (3) astral MT anchoring and behavior at the cell cortex. Aberrant regulation of astral MTs has been shown to correlate with spindle misorientation. For example, centrosomal proteins regulating γTuRC-mediated nucleation of MTs and MAPs controlling MT stability have been shown to regulate spindle orientation in their capacity of modulating MT dynamics. Despite the emerging insight into how astral MT formation is controlled to ensure proper MS orientation, the molecular mechanisms are incompletely understood (Malerod, 2018).

The multifunctional adaptor protein

date revised: 12 January 2022

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