gammaTubulin at 23C
During early embryogenesis of Drosophila, mutations in the DNA-replication checkpoint lead to chromosome-segregation failures. These segregation failures are associated with the assembly of an anastral microtubule spindle, a mitosis-specific loss of centrosome function, and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. The DNA-replication inhibitor aphidicolin and DNA-damaging agents trigger identical mitotic defects in wild-type embryos, indicating that centrosome inactivation is a checkpoint-independent and mitosis-specific response to damaged or incompletely replicated DNA. It is proposed that centrosome inactivation is part of a damage-control system that blocks chromosome segregation when replication/damage checkpoint control fails (Sibon, 2000).
It has been proposed that a component of the DNA-synthesis machinery becomes rate-limiting during the later syncytial divisions, and that activation of the DNA-replication checkpoint thus produces the increases in embryonic cell-cycle length that characterize these divisions. This model predicts that checkpoint-mutant embryos will initiate mitosis before completing DNA replication specifically during the later syncytial divisions, when the mitotic defects first appear. It was therefore speculated that the centrosome defects and segregation failures in checkpoint-mutant embryos resulted from incomplete DNA replication at mitosis, and that the checkpoint pathway is not specifically required to maintain centrosome function. In wild-type embryos, the DNA-replication inhibitor aphidicolin triggers cell-cycle delays, but mitosis is eventually initiated before S phase is completed. Thus, to test the hypothesis, mitosis was examined following treatment of wild-type embryos with aphidicolin. Under these conditions, gamma-tubulin, and the gamma-tubulin ring complex components Dgrip84 and Dgrip91 are displaced from a core centrosome structure. Aphidicolin triggers mitosis-specific loss of centrosomal gamma-tubulin, Dgrip84 and Dgrip91 during all of the syncytial divisions, in both wild type and checkpoint mutants. This structural response to aphidicolin is therefore replication-checkpoint-independent and is not specific to the later syncytial divisions. This structural response to aphidicoline occurs in grp and mei-41 mutant embryos, and is therefore replication-checkpoint-independent (Sibon, 2000).
To analyse the effects of aphidicolin on spindle assembly and chromosome segregation directly, embryos were injected with a mixture of aphidicolin, rhodamine-labelled tubulin and the DNA label Oligreen and were examined by time-lapse confocal microscopy. The results showed that aphidicolin leads to loss of tubulin foci at nuclear envelope breakdown (NEB), assembly of anastral spindles, and defects in chromosome congression and segregation. Furthermore, the centrosomal foci are re-established on exit from mitosis. Thus aphidicolin treatment accurately phenocopies the mitotic defects observed in checkpoint mutants, indicating that centrosome inactivation and the associated chromosome-segregation failures in these mutants are probably triggered by the presence of incompletely replicated DNA at mitosis (Sibon, 2000).
To determine whether photodamage to DNA also triggers centrosome inactivation and chromosome-segregation defects, laser illumination was used to induce DNA damage. Bipolar spindles are established shortly after NEB, at the time that microtubules originating at the poles interact with each other and with the mitotic chromosomes, and chromosomes rapidly congress to the metaphase plate. During mitosis 12, anaphase is initiated roughly 4 min after NEB, and chromosome segregation is completed and the nuclear envelope begins to reform ~3 min later. However, when the intensity of laser illumination is increased to 90% of maximum, the centrosomal tubulin foci decrease in intensity at NEB and disorganized microtubule bundles form around the condensed chromosomes. Bipolar spindles with broad poles eventually assemble, but the chromosome arms never fully compact at the metaphase plate and chromosome segregation fails. On intense laser illumination in checkpoint mutants and in wild-type embryos, small tubulin-containing dots are often found near poles of the anastral spindles. These structures do not appear to nucleate microtubules during mitosis, but gain nucleating function on exit from mitosis. These structures thus appear to be centrosomes that are inactivated at NEB and reactivated on exit from mitosis. X-ray treatments and ultraviolet light also trigger mitosis-specific loss of centrosomal gamma-tubulin, anastral spindle assembly, and defects in chromosome congression and segregation. On the basis of these observations, it is concluded that a variety of DNA-damaging agents and replication defects can trigger mitosis-specific centrosome inactivation and mitotic chromosome-segregation defects (Sibon, 2000).
Thus it has been shown that the chromosome-segregation defects in grp and mei-41 checkpoint-mutant embryos are linked to loss of centrosome function and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. However, this does not reflect a specific requirement for grp or mei-41 in maintaining centrosome function. The alleles analysed here are null, yet the mutant embryos proceed through 11-12 normal mitotic divisions before mitotic defects are observed. In addition, replication inhibitors or DNA-damaging agents trigger cytologically identical centrosome defects in wild-type embryos. It is therefore concluded that the loss of centrosome function and mitotic failures in grp and mei-41 mutants reflect activation of a replication-checkpoint-independent response to incomplete DNA replication or DNA damage at mitosis (Sibon, 2000).
Incomplete DNA replication or DNA damage could lead to kinetochore defects, which trigger the spindle-assembly checkpoint; centrosome inactivation could be a response to activation of this checkpoint. However, the localization of gamma-tubulin to the centrosome is not significantly affected by treatment with colchicine, which triggers metaphase arrest through the spindle-assembly checkpoint pathway. In addition, centrosome inactivation does not block centromere alignment, indicating that kinetochore function is not severely compromised when the centrosomes are inactivated. Therefore, DNA-replication- and DNA-damage-associated centrosome inactivation are both independent of the replication and the spindle checkpoints, and appear to reflect the action of a new pathway that links centrosome function to the physical state of the genome (Sibon, 2000).
Mitotic spindle assembly is normally dominated by astral microtubules, which are nucleated at centrosomes. The chromosome-segregation defects in checkpoint mutants and in wild-type embryos treated with aphidicolin or DNA-damaging agents are tightly linked to loss of centrosome-organized microtubules. These observations indicate that asters may be required for spindle function. However, anastral spindles are common during female meiosis and are found in some unusual mitotic systems and cell-free extracts. In addition, spindles in Drosophila embryos mutant for cnn have severely reduced asters, and these anastral spindles may mediate chromosome segregation. Therefore, DNA replication or damage could induce centrosome inactivation and modifications to other functions that are essential for anastral spindle function (Sibon, 2000).
Besides gammaTubulin at 23C, there is a second gamma-tubulin gene in Drosophila, gammaTubulin at 37CD. The two gammaTubulins are differentially expressed during gametogenesis and development. Both gammaTubulins are present throughout embryogenesis and larval development but the two are differentially organized during oogenesis. During oogenesis, gammaTub23C
is detected at centrosomes and in the cytoplasm of mitogenic germ cells, but is not detected in germ cells following completion of mitosis. Conversely, gammaTub37CD is not detected in proliferating germ cells, but appears to accumulate later in germ cells during egg chamber development. gammaTub37CD is usually detected in the germ cell cytoplasm of stage 4-5 and older egg chambers. Enriched accumulation of gammaTub37CD is never detected at the anterior or posterior poles of the oocyte. Neither isoform is detected at the anterior or posterior poles of developing oocytes. This is surprising since gammaTubulin has been localized to microtubule organizing centers in virtually all species examined. gammaTub37CD distribution appears to be diffuse in the germ line and not localized to MTOCs. During spermatogenesis, only gammaTub23C is detected at centrosomes, where it shows cell-cycle- and differentiation-dependent organization. During the transition into the first meiotic division, gammaTub23C becomes organized as a corpuscular focus at centrioles until completion of meiosis II. During postmeiotic spermatid differention, gammaTub23C is detected first as a rod and then as a collar-like structure near the juncture of the nucleus and the elongating flagellum, but is not detected in bundles of mature sperm. The germline-specific CDC25, encoded by twine is required for the organization of gammaTub23C into corpuscular focus in spermatocytes, but not for separation of centriole pairs in M-phase or postmeiotic organization of gammaTub23C at centrioles. Following reconstitution of a canonical centrosome at fertilization, only gammaTub37CD is detected at centrosomes in syncytial embryos, but both gamma Tubulins are detected at centrosomes in cellularized embryos. The specialized pattern of gammaTub37CD expression during cellularization raises the question of whether a unique structural feature of gammaTub37CD facilitates mitosis during early cleavage division of the embryo when centrosomes must duplicat every 9-20 minutes. Colocalization of these two isoforms suggests that both contain structural features of gamma-tubulins essential for localization to centrosomes (Wilson, 1997).
Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component
of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes: gammaTUB23C and gammaTUB37C. The 37C protein can only be detected in ovaries and embryos. Antibodies against this isoform
predominantly label the centrosomes in embryos from early cleavage divisions until cycle
15, but fail to reveal any particular localization of gamma-tubulin in the developing egg
chambers. The loss of function of this gene results in female sterility and has no effect on
viability or male fertility. Early stages of oogenesis are unaffected by mutations in this
gene, as judged both by morphological criteria and by localization of reporter genes, but
the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs
laid by mutant females is also impaired. It is concluded that the expression of the 37C gamma-tubulin isoform of D. melanogaster is under strict developmental regulation and
that the organization of the female meiotic spindle requires gamma-tubulin (Tavosanis, 1997).
In the Drosophila oocyte, meiosis is arrested in the first division of metaphase, when a tapered spindle aligned parallel to the egg surface forms. The chromosomes are therefore located in the cortical region near the anterior pole, whereas fusion of parental complements occurs in the inner ooplasm. How does the female pronucleus reach the interior of the egg? The second meiotic spindles are arranged in tandem, end to end, and disposed perpendicular to the longitudinal axis of the egg with the innermost spindle carrying the female pronucleus. This pattern of spindle organization is probably involved in the migration of the female pronucleus deeper into the egg near the cytoplasmic domain of the male pronucleus. The precise time at which the mitotic spindle of Drosophila changes orientation is unknown. However, spindle rotation from a position parallel to the egg surface to a radial orientation presumably occurs during or shortly after the oocyte passes through the oviduct. How spindle orientation is achieved and maintained during meiosis is an intriguing question. Microtubules linking spindle poles to the oocyte surface have been implicted in the rotation and anchoring of the meiotic apparatus in Xenopus oocytes and in other organisms, but this does not seem to be the case in the Drosophila oocyte, since the meiotic spindles lack astral microtubels. However, the observation that a transient array of microtubules links the meiotic apparatus to discrete subcortical foci suggests that in Drosophila the orientation of the spindle also requires a functional interaction between the spindle and the oocyte cortex (Riparbelli, 1996 and references).
The microtubule array of mitosis II observed between the twin spindles at metaphase, anaphase and telophase might be an intermediate between the anastral poles of the meiotic I spindles and the astral poles of the mitotic spindles in early embryos. A complex pathway of spindle assembly takes place during resumption of meiosis at fertilization, consisting of a transient array of microtubules radiating from the equatorial region of the spindle toward discrete foci in the egg cortex. A monastral array of microtubules is observed between twin metaphase II spindles in fertilized eggs. These microtubules originating from disc-shaped material stain with Rb188 antibody specific for an antigen asssociated with the centrosome of Drosophila embryos (DMAP190 or CP190). Therefore, the Drosophila egg contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubues in a monastral array after activation, but are unable to organize bipolar spindles (Riparbelli, 1996).
The meiosis II spindle of Drosophila oocytes is distinctive in structure, consisting of two tandem spindles with anastral distal poles and an aster-associated spindle pole body between the central poles. Assembly of the
anastral:astral meiosis II spindle occurs by reorganization of the meiosis I spindle, without breakdown of the meiosis I
spindle. The unusual disc- or ring-shaped central spindle pole body forms de novo in the center of the elongated
meiosis I spindle, followed by formation of the central spindle poles. gamma-Tubulin transiently localizes to the
central spindle pole body, implying that the body acts as a microtubule nucleating center for assembly of the central
poles. The first step in formation of the central pole body is the appearance of puckers in the center of the the meiosis I spindle, followed by the pinching out from the spindle of a disc or ring of microtubules that becomes the central pole body. The manner in which the central spindle pole body forms suggests the involvement of a microtubule motor. If so, the motor involved is likely to be different from Ncd (Nonclaret disjunctional), since loss of Ncd function does not seem to prevent its formation. Following the formation of the central spindle pole body, the microtubules arrayed to either side of the central body narrow into poles, forming the mature meiosis II spindle. The central poles become more tapered during progression through meiosis II, and the central spindle pole body also changes in morphology: the disc or ring becomes asterlike, then enlarges into a ring that lies between the two central telophase II nuclei (Endow, 1998).
Localization of gamma-tubulin to the meiosis II spindle is dependent on the microtubule motor protein,
Ncd. Absence of Ncd results in loss of gamma-tubulin localization to the spindle and
destabilization of microtubules in the central region of the spindle. Likewise, during meiosis I, the minus-end motility of Ncd and its crosslinking activity are probably needed to focus microtubules into spindle poles for the correct functioning of meiosis I. Assembly of the anastral:astral meiosis II spindle
probably involves rapid reassortment of microtubule plus and minus ends in the center of the meiosis I spindle. This
can be accounted for by a model that also accounts for the loss of gamma-tubulin localization to the spindle and
destabilization of microtubules in the absence of Ncd (Endow, 1998).
A model for assembly of the Drosophila oocyte meiosis II spindle is suggested: gamma-Tubulin is first recruited or relocalized, possibly as gamma-TuRC, to the midbody of the meiosis I spindle, where it functions to nucleate microtubules for formation of the meiosis II central spondle poles. The loss of gamma-tubulin localization to the spindle in the absence of Ncd suggests that the Ncd motor serves to recruit or anchor gamma-tubulin to the center of the spindle. The Ncd motor would then stabilize newly nucleated microtubule minus ends and focus the microtubules into poles. The unstabilized plus ends of the microtubules in the center of the spindle (remaining from meiosis I) would undergo rapid depolymerization as a consequence of dynamic instability. Stabilization of the newly nucleated microtubule minus ends and depolymerization of the plus ends would cause a rapid sorting out of the microtubules in the center of the meiosis I spindle, replacing microtubule plus ends with minus ends. The distal poles of the meiosis II spindle would be retained from the meiosis I spindle and maintained by the same forces that originally formed them: the crosslinking activity and minus-end movement of Ncd along spindle microtubules (Endow, 1998).
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. dd4S 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
(gammaTub23CPI). 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 dd4S. 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
dd4S 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
gammaTub23CPI 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
gammaTub23CPI 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 gammaTub23CPI
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).
Molecular motors transport the axis-determining mRNAs oskar,
bicoid and gurken along microtubules (MTs) in the
Drosophila oocyte. However, it remains unclear how the underlying MT
network is organized and how this transport takes place. A
centriole-containing centrosome has been detected close to the oocyte nucleus. Remarkably, the
centrosomal components, gamma-tubulin and Drosophila
Pericentrin-like protein also strongly accumulate at the periphery of this
nucleus. MT polymerization after cold-induced disassembly in wild type and in
gurken mutants suggests that in the oocyte the centrosome-nucleus
complex is an active center of MT polymerization. The
MT network comprises two perpendicular MT subsets that undergo dynamic
rearrangements during oogenesis. This MT reorganization parallels the
successive steps in localization of gurken and oskar mRNAs.
It is proposed that in addition to a highly polarized microtubule scaffold
specified by the cortex oocyte, the repositioning of the nucleus and its
tightly associated centrosome could control MT reorganization and, hence,
oocyte polarization (Januschke, 2006).
Both the nature and the localization of the MTOC beyond stage 6 of
Drosophila oogenesis have not yet been clarified. Up to stage 6,
gamma-tubulin has been shown to closely associate with the nucleus at the
posterior of the oocyte. In addition, electron microscopy studies have
demonstrated the presence of centrioles close to the oocyte nucleus up to
stage 4. Thus, until stage 6, the centrosome associates with the
nucleus at the posterior of the oocyte. In Drosophila females,
meiosis takes place in the absence of centrosomes. It has therefore been speculated that, at stage 6, centrosome organization changes, involving the disappearance of centrioles and the generation of MTs from a diffuse organizing center. To better understand this process, the distribution of gamma-tubulin in the
oocyte was re-investigated. Before repolarization of the MT cytoskeleton, it was found that gammaTub23C and gammaTub37C localize in a layer around the nucleus, with an enrichment at the posterior pole of the oocyte. This is in agreement with the location of the MTOC at this stage. After repolarization of the MT cytoskeleton, both gamma-tubulin isoforms remain located in a perinuclear manner. Interestingly,
gammaTub37C, but not gammaTub23C, labels a small body close to the oocyte
nucleus. In addition, gammaTub37C and gammaTub23C also exhibit differential
expression patterns in embryos: gammaTub37C is located with the centrosomes
of mitotic cells, whereas gammaTub23C is not. Thus, gamma-tubulin is distributed in close association with the nucleus periphery and possibly on a centrosome-like structure. Pericentrin/AKAP450 is another major component of the centrosome. Green
fluorescence protein (GFP) fusion of the C-terminal part of
Pericentrin/AKAP450 and its Drosophila homolog pericentrin-like
protein (D-PLP) have been shown to localize to the centrosomes respectively in
cultured human cells, Drosophila embryos and spermatocytes. Using
the UAS/Gal4 system, GFP-cter-D-PLP was specifically expressed in the germline
and a bright dot was detected in the vicinity of the nucleus before and after
nuclear migration. GFP-cter-D-PLP was also detected in all germline nuclei, as has been observed previously. From stage 7 onward, the bright dot remained in the immediate vicinity of the oocyte nucleus (<1 µm distance). Furthermore, both GFP-cter-D-PLP and gammaTub37C co-localize to this discrete body, indicating that this structure could correspond to a centrosome. In G2 centriole, tubulin is highly polyglutamylated. The
ID5 antibody labels basal bodies and centrioles in several species. Using
this antibody, a dot was detected close to the nucleus throughout oogenesis
that remained detectable up to stage 10A. This suggests that
the dot represents a centriole-containing centrosome. Indeed, using electron
microscopy, two to possibly four centrioles were clearly detected closely associated with the nucleus in stage 9 oocytes. This demonstrates the existence of centrioles associated with the nucleus at least up to stage 9. MT fibers emanating from those centrioles could not be unambiguously detected. Then the link between centrosome and nucleus was examined using colchicine. In flies fed with colchicine, MTs in the germline were completely depolymerized, and the oocyte nucleus was mispositioned. In the oocyte, it was observed that the nucleus and the centrosome were significantly separated, the distance between them increasing during oocyte growth. In a few cases, it was noticed that the nucleus could reach the anterior cortex without the centrosome; however, a centrosome was never observed at the anterior without the nucleus. It is concluded that the close localization of the centriole-containing centrosome to the nucleus depends on MTs (Januschke, 2006).
The structure of the MT network during mid-oogenesis is dynamic. At stage
7, MTs are visible as a mesh at the anterior cortex. Later, at stage 10, MT
bundles are observed that promote cytoplasmic streaming. In-between MT distribution has been described as an AP gradient.
However, high-resolution images of oocyte MTs are lacking. Therefore, a protocol frequently used to
increase the detection of the MT cytoskeleton in cell culture was
modified for the Drosophila egg chamber to characterize
MT organization in the oocyte during the crucial period in which bcd,
grk and osk mRNAs are localized. MTs were detected throughout
oogenesis using alpha-Tubulin but also with a Kinesin heavy chain antibody
(alpha-Khc), which revealed the MT array and its complexity in unprecedented
definition. It was noticed that the range of detected details was increased and more reproducible with alpha-Khc antibody. To control the specificity of Khc detection, germline and follicle cell mutant clones were
generated homozygous for
khc7.288. In such mutant cells, no Khc was detected, indicating
that the detection is specific. Labeling with antibodies directed against
aromatic C-terminal amino acid residues (Tyr or Phe) of alpha-tubulin and against Khc largely overlapped. This confirmed
that the structures revealed by Khc were MTs. A Khc fraction was also detected
at the posterior of the oocyte. That
Khc accumulates along MTs may be due to permeabilization before fixation,
which could cause rigor binding of Khc to MTs. This
detection procedure may also permit the extraction of a soluble pool of Khc
and reveal the remaining fraction distributed along the MTs. With this
detection procedure, Khc revealed by Kinesin-ßgal exhibited
a more restricted distribution compared with alpha-Khc antibody. This is probably
due to the substitution of the C-terminal part of Khc by the
ß-galactosidase in the reporter construct, impairing the recycling of the
chimeric Kinesin motor leading to its accumulation exclusively at the
posterior. With this detection method, the MT minus-end
marker, Nod-ßgal, was detected in the antero-dorsal corner above the
oocyte nucleus as well as in the opposite antero-ventral corner. Moreover, localized determinants such as Osk and Grk were
correctly positioned in the oocyte (Januschke, 2006).
To confirm that the detection method does not alter MT organization, MT distribution was analyzed in follicle cells, which should be sensitive to the
extraction procedure, since they are more directly exposed than the oocyte. MT distribution in different follicle cell types was unchanged, when comparing living and fixed egg chambers. The main body follicle cell MTs seemed unchanged. Main body follicle cell MTs have been shown to be highly stable, and might therefore reflect the sensitivity of the protocol with limitations.
Nevertheless, stretched follicle cells showed strikingly similar MT patterns
in living and fixed conditions as well. Apicalbasal
polarity was not affected in follicle cells, as demonstrated by the correct
apical localization of atypical protein kinase C. Importantly, the
MT distribution of living egg chambers expressing GFP-alpha-Tubulin at stage
7 and stage 9 was similar to the
one observed using anti-alpha-Tubulin and Khc antibodies. Therefore it
seems that the fixation conditions preserve the wild-type MT organization and
that Khc can be suitable to label bulk MTs (Januschke, 2006).
When fixed wild-type oocytes were analyzed by confocal microscopy, MT
organization in the oocyte appeared unchanged from stage 2 to
stage 6. With stage 7, MT organization was modified and two
MT subsets became apparent. This organization was more evident at stage 8. A first subset consisted of cortical MTs oriented
along the dorso-ventral (DV) axis parallel to the oocyte nurse cell border,
and juxtaposed to the lateral cortices, wrapping the oocyte from stage 7 to 9. At
least some MT bundles of this subset could be traced back to the oocyte
nucleus. The DV orientation of MT bundles, depicted as black fibers in the
schematic representations, was highly reproducible for all stages and persisted
throughout mid-oogenesis (Januschke, 2006).
A second MT subset was present
in the center of the oocyte. Although there was some variability in the
patterns observed, it was found that each developmental stage showed a
characteristic MT distribution. During stage 6, MTs
from this subset were cortical and extended from the nucleus at the posterior
to the anterior cortex, compact
bundles of MTs formed a circle-like structure resembling a diaphragm. This
subset was formed by long MT bundles that extended (once or more) along the
entire cortex. By stage 8, the oocyte had considerably grown
and individual MT bundles were therefore easier to track. MT bundles emanated
from the anterior and the nucleus to point toward the posterior. MTs extended
again along the entire cortex, after which they turned to the central
cytoplasm. This, in turn, generated free MT (plus) ends in the center of the oocyte. By stage 9, the central MT network was clearly oriented along
the oocyte AP axis. One or two thick MT bundles extended from the anterior,
pointing toward the posterior pole. These bundles formed a structure
resembling a horseshoe, with its open side facing the posterior.
Importantly, both subsets could also be detected in living egg chambers, as
shown for the DV subset and the AP subset. Thus, MTs show strong rearrangements throughout mid-oogenesis, which results in two perpendicular MT arrays reflecting the two axes of the oocyte (Januschke, 2006).
An ex-vivo assay was developed to localize MT nucleation sites by dissecting
ovaries and placing them on ice for 30 minutes. This treatment resulted in
complete depolymerization of MTs. When allowed to recover at 25°C for 30 minutes, MT distribution could be re-established to the wild-type situation, in which both the cortical and the central subsets of MTs were detectable. gamma-Tubulin distribution was not affected by cold-induced MT depolymerization. When short periods of regrowth were analyzed, MT nucleation appeared limited to the close vicinity of the nucleus and was often asymmetric, suggesting a
centrosome-associated nucleation activity. MT regrowth appeared to be
stepwise, since after 15 minutes only the DV cortical subset was established. MTs
clustered around the oocyte nucleus and aligned along the cortex in the DV
direction. The
cortical location of these fibers was clearly revealed by the presence of
Khc-positive dots at either the dorsal or the ventral side. This indicates that
the DV MT subset is the first to regrow. The regrowth experiment was repeated
using colchicine. After the drug was washed out, MT repolymerization was
observed at the oocyte nucleus. Taken together, these results
indicate that, at least with the detection method that was used, the oocyte nucleus and its immediate surroundings have the capacity to nucleate MTs (Januschke, 2006).
To test whether the centrosome-nucleus complex could direct the
repolarization of the MT network, how MTs distribute in
grk mutant oocytes was examined. In this mutant, the nucleus frequently remains at the posterior of the oocyte due to a failure in the signaling cascade that induces the repolarization of the cytoskeleton. In
grk mutant oocytes similar in size to wild-type stage 8, the MT
distribution was dramatically affected. Specifically, MT
organization appeared completely reversed compared with wild type, in which
the nucleus is at the anterior and MT plus-ends are located toward the
posterior at stage 8. In slightly
older oocytes, MTs remain stretched out along the cortex from the posterior
toward the anterior, where they fold back to the center of the oocyte. MT ends in the center are most probably plus-ends, since the pool of Khc (localized at the
posterior of wild-type oocytes, co-localizes with Kinesin-ßGal to the center of the oocyte, between the flanking MT ends. Interestingly, MT distribution in grk oocytes was strikingly similar to MTs of wild-type egg chambers before the migration of the oocyte nucleus.
Likewise, the centrosome, as revealed by gamma-tubulin, which is found at
the posterior of stage 6 wild-type oocytes, stays at the posterior in
grk mutants.
Thus, in grk mutants, distribution of MT and MTOC seemed similar to
their distribution in wild-type stage 6 (Januschke, 2006).
grk mutant oocytes, having mispositioned nuclei, provide an ideal
basis to test the MT nucleating capacity of the centrosome-nucleus complex
using the cold-shock assay. After cold-shock treatment of grk
oocytes, complete MT depolymerization was checked for. As in the wild type,
during the initial period of recovery at 25°C, MT polymerization took place only in the immediate vicinity of the mispositioned oocyte nucleus. Therefore, as in wild-type oocytes, MT nucleation is often asymmetric and restricted to the area surrounding the nucleus. This result strengthens the possibility that the centrosome-nucleus complex is an active MTOC (Januschke, 2006).
Thus, in the Drosophila oocyte a centriole-containing centrosome is present in
close association with the nucleus, which itself is covered by PCM components
until late in oogenesis. In addition, MTs can nucleate from this
centrosome-nucleus complex. The MTs appear to form two orthogonal MT
populations that develop through several steps during mid-oogenesis. It is
proposed that the migration of the nucleus in the oocyte could control the
reorganization of the MT network (Januschke, 2006).
In region 2 of the germarium, nurse cell centrosomes migrate toward the
oocyte. Later, in region 3, these centriole-containing centrosomes become
located as an aggregate between the oocyte nucleus and the follicle cell
border. Pericentriolar material closely associated with the oocyte
nucleus can be clearly detected until stage 6 with several centrosomal
markers, such as gamma-tubulin, Centrosomin and D-Tacc. From stage 4 onward, the fate of the centriole cluster has been unknown. This study shows that both gammaTub37C and
gammaTub23C are localized in a perinuclear manner throughout oogenesis.
gammaTub37C highlights a discrete body close to the nucleus. This body is
similarly detected by the centrosomal marker D-PLP and by a specific antibody
for polyglutamylated Tubulin, which detects centrioles. Consistent with this,
two to possibly four centrioles were detected in the immediate vicinity of
the nucleus in stage 9 oocytes. This result demonstrates that at least until
stage 9, a centriole-containing centrosome is present in the oocyte.
Currently, it is not known whether they are still present at the onset of
meiosis I during stage 13, since it has previously been proposed that the meiotic
spindle is achieved without centrosomes. During skeletal muscle morphogenesis, myotube centrosomes
dissociate from their nuclei, centrioles disappear and the centrosomal matrix
is redistributed to the nucleus periphery.
Similarly, during oogenesis, centrioles from nurse cell centrosomes may
disappear. However, their pericentriolar material may relocate to the oocyte
nucleus periphery. This would explain the specific enrichment of the oocyte
nucleus with perinuclear MTOC material. The only centrosome remaining
associated with a nucleus is that of the oocyte. Furthermore, the structure of
this centrosome remains intact. It is concluded that the four centrioles found
close to the nucleus in stage 9 may correspond to the initial oocyte
centrosome in the duplication phase observed in G2 (Januschke, 2006).
In Drosophila, gammaTubulin is required for the structure as well as the function of microtubule
organizing centres (MTOCs). This conclusion is based on the identification and phenotypic characterization of a
mutant allele of the gamma-tubulin gene located at region 23C of the polytene chromosome map. This mutation, termed gamma-tub23CPl, is caused by the insertion of a P-element within the 5' untranslated leader
of the gamma-tubulin transcript. Northern and Western analysis show that gamma-tub23CPl is either a null or a
very severe hypomorph as no gamma-tubulin mRNA or protein can be detected in mutant individuals.
Visualization of DNA, MTOCs and microtubules by confocal laser scanning microscopy of cells from individuals
homozygous for gamma-tub23CPl reveals a series of phenotypic abnormalities. Some of these are similar to those
observed after disruption of gamma-tubulin function in other organisms, including mitotic arrest and a dramatic
decrease in the number of microtubules. Mutation in this gene also results
in highly abnormal MTOCs showing a variety of shapes and sizes never observed in wild type cells.
These results show that gammaTubulin is required for both structural and functional roles in the MTOCs (Sunkel, 1995).
Differentiation of the Drosophila oocyte takes place in a cyst
of 16 interconnected germ cells and is dependent on
a network of microtubules that becomes polarized as
differentiation progresses (polarization). How the microtubule network polarizes
was investigated using a GFP-tubulin construct that allows germ-cell
microtubules to be visualized with greater sensitivity than
in previous studies. Unexpectedly, microtubules are seen to
associate with the fusome, an asymmetric germline-specific
organelle, which elaborates as cysts form and undergoes
complex changes during cyst polarization. This fusome-microtubule
association occurs periodically during late
interphases of cyst divisions and then continuously in 16-cell
cysts that have entered meiotic prophase. As meiotic cysts
move through the germarium, microtubule minus ends
progressively focus towards the center of the fusome, as
visualized using a NOD-lacZ marker. During this same
period, discrete foci rich in gamma tubulin that very probably
correspond to migrating cystocyte centrosomes also
associate with the fusome, first on the fusome arms and then
in its center, subsequently moving into the differentiating
oocyte. The fusome is required for this complex process,
because microtubule network organization and polarization
are disrupted in hts1 mutant cysts, which lack fusomes. These
results suggest that the fusome, a specialized membrane-skeletal
structure, that arises in early germ cells, plays a
crucial role in polarizing 16-cell cysts, at least in part by
interacting with microtubules and centrosomes (Grieder, 2000).
Polarity within germline cysts arises at two distinct times. First,
polarizing forces build the asymmetric structure of the cyst in
region 1 and are dependent on the fusome. Later, polarizing
forces operate in fully formed 16-cell cysts to re-organize and
polarize the MT network towards the differentiating oocyte.
Since the oocyte is invariably one of the four-ring canal cells,
early asymmetries must be preserved and re-used within
polarizing 16-cell cysts; however, the mechanisms responsible
for preserving and interpreting early polarity information have
remained unclear. The fusome is required
to organize a complex and dynamic MT network both in
dividing cysts of region 1 and also in polarizing 16-cell cysts.
The experiments reported here strongly suggest that a
persistent asymmetry within the fusome stores early polarity
information for use in fully formed cysts. Moreover, this
polarity is likely read out through an action of the fusome on
microtubule organization. In the absence of a fusome,
microtubule polarity in meiotic cysts is abolished and oocytes
do not form (Grieder, 2000).
The requirement of a fusome to organize MTs and polarize 16-cell cysts provides the strongest argument that the associations observed between MTs and the fusome are functionally
important. However, it could be argued that the MT
organization in hts mutants is disrupted by a fusome-independent
mechanism. A processed fragment of Hts protein
is incorporated into maturing ring canals in region 2b, raising the possibility that ring canals,
rather than the fusome, mediate MT polarization. This interpretation, however, is not supported by experimental observations. The
MT network associates with the fusome and not the ring canals,
and this association is detected in late interphase of cyst
divisions and in meiotic cysts in region 2a, long before
Hts proteins are incorporated into ring canals. All these
associations, not just those in region 2b, are disrupted in hts
mutants. Consequently, the fusome rather than the ring canals
is responsible for organizing MTs during early germ-cell
development (Grieder, 2000).
Genetic studies of three molecules that mediate transport
along microtubules provide further evidence for a connection
to the fusome. Mutations in cytoplasmic DHC64C disrupt cyst
production and block production of a normally structured
fusome, at least in part by disrupting the normal orientation of
the mitotic spindle with the fusome during cyst divisions. Unfortunately, it has remained
unclear whether DHC64C is localized to the centrosomes and
the fusome during prophase of meiotic cysts. It is, however,
tempting to speculate that DHC64C plays a critical role in
organizing the MTs of polarizing cysts, as there is a genetic
requirement for DHC64C in oocyte formation
and DHC64C staining is lost in hts mutant cysts. In addition, Drosophila Lissencephaly-1 is involved in MT
dynamics. LIS1 mutants have defects not only in MT
organization but also in fusome formation. Finally, mutations in the kinesin-like
protein KLP61F interfere with the production of a normal
fusome in Drosophila males. Whether these
last two proteins have a specific role during polarization or only during cyst formation remains unclear (Grieder, 2000).
These observations reveal that a novel mechanism, directed
centrosome migration, is likely to be involved in cyst
polarization. Foci rich in gamma tubulin move onto and along the
fusome arms in region 2a and 2b cysts, indicating that the
nurse-cell centrosomes migrate along the fusome on their way
to the oocyte. The total number of gamma tubulin foci in region 2a
cysts range between 13 and 20, consistent with the number
expected if most of the cystocyte centrosomes were labeled.
The developmental changes in the timing and the approximate
number of gamma-tubulin foci are indistinguishable from the
behavior of migrating centrioles. The association of the fusome with this process of
directed movement provides further evidence that the fusome
is itself a polarized structure, not only during its formation in
region 1, but also in region 2 cysts. It will be important to look for
specific molecules that link centrosomes to the fusome and
move them in a directional manner in response to fusome
polarity (Grieder, 2000).
Previously, only the movement of centrioles, not
centrosomes, was reported.
It has been suggested that the migrating centrioles are inactive and
break down in the oocyte so that centriolar constituents can be
re-used in the early embryo. The gamma-tubulin staining observed
argues that the migrating centrioles are accompanied by peri-centriolar
material and can actively nucleate microtubules. The
preferential accumulation of active centrosomes in the central
region of the fusome would probably contribute to polarizing
the MT network. Later movements of the centrosomes probably
contribute to subsequent stages of polarization. These include
the focusing of the MT minus ends within a single cyst cell, and
the movement of MTs to the posterior of the oocyte. The
existence of 13-20 actively migrating centrosomes is not
consistent with the suggestion that MTOC formation and oocyte
determination are controlled by the selective inheritance of a
single active centrosome (Grieder, 2000 and references therein).
At least some of the microtubule organizing activity of the
fusome is not due to associated centrosomes. During late
interphase of the cystocyte divisions, the main part of the
fusome strongly interacts with microtubules. Many MTs and/or
MT bundles exit the fusome at this time, and it appears unlikely
from their number and distribution that associated centrioles
could account for all this activity. Even though many discrete
gamma-tubulin spots/centrosomes were localized on or close to the
fusome arms in region 2a, the overall MT polarity is not
uniformly oriented towards the fusome, based on the NOD-lacZ
staining. Consequently, the fusome may transiently acquire the ability to bind or bundle MTs, rather than nucleating them (Grieder, 2000).
At other times, the fusome might interact directly with MT
minus ends. Fusomal material may acquire the ability to
nucleate MT minus ends by associating with peri-centriolar
material. The fusome in polarizing cysts appears most likely to
exhibit such an activity, since MT-fusome interaction during
polarization involves MT minus end interactions. Alternatively,
the fusome might interact with motor proteins that bridge it to
MTs; these proteins would specifically interact with minus ends. Polarization of
the MT network into the differentiating oocyte could involve
both minus end interaction as well as bundling. Both of these
mechanisms would probably be amplified through MT-MT
interactions outside the fusome (Grieder, 2000).
Previously, it has been unclear when and if the fusome
contained polymerized microtubules. The retention of such
MTs has been proposed to store polarity information from the
fusome for later use in developing 16-cell cysts. GFP-alpha-tubulin staining does reveal MTs that run parallel to the fusome in region 2b cysts. However, there
is no evidence for such MTs in aligning cysts within region 2a,
or within the main body of the fusome in early interphase of the
cystocyte divisions. Consequently, polymerized MTs within
the fusome that persist from the time of cyst formation do not
appear to be the basis of the fusomal polarity (Grieder, 2000).
The use of a highly sensitive GFP-alpha-tubulin fusion protein
reveals a more detailed picture than previously available of
the dynamic process of MT polarization in developing 16-cell
cysts. At the beginning of the polarization process in oblique
meiotic cysts, MTs extend equally into all cystocytes and are
embedded in the fusome. Subsequently, MT minus ends coalesce
towards the central region of the fusome, which resides within
both four-ring canal cells and their immediate neighbors.
Gradually, the MT network continues to polarize until it
becomes clearly focused in a single cell, the future oocyte, late
in region 2b. Thus, an organized microtubule cytoskeleton does
not arise spontaneously within region 2a cysts in response to
the appearance of a new MTOC in one of the cyst cells. Rather,
the MT network polarizes progressively over an extended
period of time and the new MTOC develops gradually (Grieder, 2000).
These observations clarify the likely relationship between cyst
polarization and the asymmetric accumulation of specific
molecules. The exact stage at which specific RNAs and
proteins accumulate has remained unclear in previous studies,
because reagents differing in sensitivity were often used. Only after
a significant fraction of the MT minus ends become
concentrated would mRNAs and proteins whose transport is
MT dependent be able to accumulate in their vicinity. MT
minus ends begin to cluster in mid- to late-region 2a cysts; it is
proposed that this event determines when specific molecules
begin to accumulate unequally. This is consistent with the
observation that orb, hts and probably other mRNAs become
clearly localized within a small region of the cyst as early as
the middle of region 2a in some germaria (Grieder, 2000).
The origin of cellular asymmetry is being studied in diverse
systems, including the formation of yeast buds, the
development of embryonic axes in C. elegans and Drosophila
embryos, the segregation of Drosophila neuroblasts and the
polarization of epithelial layers. In all these systems, there is evidence that the
microtubule cytoskeleton interacts with other cytoskeletal
components, particularly actin, to orient divisions and mediate
intracellular asymmetry. For example, localization of the MT-orienting
KAR9 protein in yeast depends on actin. The asymmetric distribution of proteins after the first division of the C. elegans embryo requires actin, and some
localized gene products such as PAR1 appear to act on
microtubules. In at least one case, the requirement for actin in
spindle orientation exists during only a portion of the cell cycle.
The results presented here indicate that Drosophila germline cysts provide
another system where the development of polarity depends on
interactions between the microtubule cytoskeleton and another
cytoskeletal system, the fusome. The fusome arises early in
germ cell development at the time germ cells migrate toward
the gonadal mesoderm, and subsequently is often located near the centrioles. Interactions, between the fusome and the MT cytoskeleton may take place
in some form during much of germ-cell development, and be
elaborated as cysts form and polarize. Further study of the
molecular basis of fusome-microtubule interactions is likely to advance an understanding of many aspects of cell and tissue polarization (Grieder, 2000).
To assess the role of gamma-tubulin in spindle assembly in vivo, meiosis progression has been followed by immunofluorescence and time-lapse video microscopy in gammaTub23CPI mutant spermatocytes. Centrosomes associate with large numbers of astral microtubules even though gamma-tubulin is severely depleted; bipolar meiotic spindles are never assembled; and later in meiosis, the microtubules get organized into a conical structure that is never observed in wild-type cells. Several lines of evidence suggest that these cones may be related to wild-type central spindles: (1) they are assembled midway through meiosis and elongate during anaphase; (2) they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type spindle midzone; (3) Klp3A and Polo, two markers of the wild-type central spindle are also found around the pointed end of the mutant cones, and (4) ectopic cytokinesis furrows are often formed at the distal end of the cone. These results suggest that microtubule polymerization or stabilization from the centrosome may be possible in a gamma-tubulin-independent manner in Drosophila spermatocytes. However, gamma-tubulin seems to be essential for spindle assembly in these cells. Finally, these results show that at least part of the central spindle and constriction-ring assembly machinery can operate on microtubule bundles that are not organized as bipolar spindles (Sampaio, 2001).
Two gamma-tubulin isoforms have been identified in Drosophila, gammaTub37C and gammaTub23C. gammaTub37C is largely restricted to the female germ line and early stages of embryogenesis. The gammaTUB23C isoform, in contrast, is expressed in a variety of tissues in both sexes and is the only isoform present in testes. Genetic, biochemical, and cytological data suggest that the gammaTub23CPI mutant allele leads to a very severe loss of function of the gammaTub23C gene and thus to a severe reduction of the levels of gamma-tubulin in mutant testes (Sampaio, 2001).
At the onset of meiosis, the two asters segregate normally in gammaTub23CPI mutant spermatocytes, but soon afterwards, they collapse back together again. As meiosis proceeds, the normal figures found in control cells are never observed in gammaTub23CPI spermatocytes. Instead, the microtubules form cone-shaped structures, one per cell, with a pointed end and the two asters fused or very close to each other at the base. Such figures have not been reported before in other meiotic mutants in Drosophila. gammaTub23CPI early spermatid cysts contain only 16 cells, and each spermatid has several nuclei of different sizes associated with a single large nebenkern, revealing the failure of the two meiotic divisions. Around 75% of these cysts contain small anuclear and anastral cell fragments that carry a small nebenkern, suggesting that some highly asymmetric cytokinesis takes place in these cells (Sampaio, 2001).
To further investigate the organization of microtubules in gammaTub23CPI meiotic spermatocytes, fixed preparations were stained with antibodies against alpha-tubulin. At prophase, a pair of well-defined astral arrays can be observed both in wild-type and gammaTub23CPI mutant spermatocytes. By the time of prometaphase/metaphase, a considerable number of microtubules are present in gammaTub23CPI mutant spermatocytes, but they only form a disordered mesh that bears no resemblance to the bipolar spindles found at this stage in control spermatocytes. As meiosis proceeds, the microtubules found in the mutant spermatocytes are organized into cones. With very rare exceptions, the chromosomes and centrosomes are located at the base of the cone (Sampaio, 2001).
The abundance of microtubules in gammaTub23CPI spermatocytes is quite remarkable given the very low gamma-tubulin levels observed in these cells. If the leaky function provided by this mutant allele is able to sustain the observed levels of microtubule polymerization, gamma-tubulin must be present in vast excess in wild-type spermatocytes. Alternatively, microtubule polymerization and stabilization at the centrosome may not be completely dependent upon gamma-tubulin in these cells. This interpretation is also supported by previous work carried out in Drosophila, Caenorhabditis elegans, and Schizosaccharomyces pombe (Sampaio, 2001).
However, despite the presence of a significant number of microtubules, bipolar spindles are never assembled in gammaTub23CPI mutant spermatocytes. This is a surprising result given that spindle self-assembly through a centrosome-independent pathway has been reported in several experimental models, including Drosophila neuroblasts and spermatocytes, in vitro. The reasons for this failure to organize the meiotic spindle are not understood. A simple explanation could be the presence of impaired, but partially functional, centrosomes that may still function as the major MTOCs (microtubule organizing centers) in these cells, thus overriding the organizing activity of the acentrosomal spindle assembly pathway. This hypothesis is not supported by the absence of organized spindles in gammaTub23CPI;asl double-mutant spermatocytes. However, some centrosomal function may still remain in the double mutant. In this regard, it would be very interesting to investigate the phenotype of cnn gammaTub23CPI double-mutant spermatocytes. An alternative interpretation could be that the dynamic properties of the microtubules present in gammaTub23CPI cells are such that they no longer serve as good substrates for the organizing activities of the molecular motors involved in the acentrosomal spindle assembly pathway (Sampaio, 2001).
To get a better understanding of the process of assembly of the cones found in gammaTub23CPI spermatocytes, meiosis was followed in these cells by time-lapse microscopy. At late prophase, the two mutant asters that had previously migrated apart start to approach each other, and the remains of the nuclear envelope become deformed. Similar results have been observed in C. elegans embryos in which, following gammaTub inactivation by RNAi, the two asters that had initially segregated collapse back together again. As meiosis proceeds in the mutant cells, the condensed bivalents are clearly visible, and a large number of microtubules is revealed by their association with phase-dark membranes. At this stage, most microtubules are sorted into two populations that emanate from each aster. Their distal ends can be seen moving along the cell membrane, becoming clustered at one side of the cell. Coinciding with the separation of the homolog univalents, and thus at a stage similar to anaphase, the microtubule array found in the mutant starts to elongate and continues to do so until the new nuclear envelopes are formed. Finally, this microtubule network is disassembled at the end of meiosis I. Thus, the time of organization and elongation of the cone found in gammaTub23CPI spermatocytes corresponds with the timing of organization and elongation of the wild-type central spindle (Sampaio, 2001).
Given the similarities in the dynamics of assembly and elongation of mutant cones and wild-type central spindles, the presence of common molecular components was investigated. To this end, the localization of the wild-type central spindle markers Polo and Klp3A was studied in the mutant cones. Immediately before nuclear envelope breakdown, the V-shaped centrosomes characteristic of these cells are strongly labeled by a functional GFP-Polo fusion that, at this stage, also very distinctively labels the nucleolus. Two pairs of centrosomes can be seen at nearly opposite sides of the nucleus, both in wild-type and in gammaTub23CPI mutant spermatocytes. However, as meiosis proceeds in the mutant, the centrosomes get closer to each other and the spindle fails to become organized. When the microtubule cone is formed, the GFP-Polo fusion strongly accumulates at the pointed end, just as it does in the wild-type spindle midzone. It also labels the centrosomes in both wild-type and mutant cells. During cytokinesis, GFP-Polo colocalizes with the constriction ring in the wild-type. In those instances in which highly asymmetric cytokinesis take place in the mutant cells, the GFP-Polo fusion is localized near the furrow. Similar results were obtained with Klp3A. In wild-type spermatocytes, the kinesin-like protein Klp3A accumulates in the central spindle midzone. In gammaTub23CPI mutant spermatocytes, Klp3A is found at the pointed end of the cone. Therefore, at least part of the machinery involved in the assembly of the constriction ring may also be used to organize the pointed end of the cone (Sampaio, 2001).
Therefore, the cone-shaped microtubule arrays found in gammaTub23CPI mutant spermatocytes bear some striking similarities to wild-type central spindles: (1) they are assembled midway through meiosis and elongate soon afterwards, just as wild-type central spindles do during anaphase B; (2) they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type central spindle during telophase; (3) Klp3A and Polo, two of the proteins localized in the wild-type central spindle midzone that are required for the recruitment of several components of the contractile ring, are also found around the distal pointed end of the mutant cones. Also, highly asymmetric cytokinesis can occur within gammaTub23CPI mutant cells at the distal end of the cone (Sampaio, 2001).
In summary, the depletion of gamma-tubulin caused by the gammaTub23CPI mutant allele does not prevent microtubule polymerization or stabilization, abolishes spindle assembly, and allows for the activity of the mechanisms that organize central spindles (Sampaio, 2001).
The assembly of the mitotic spindle after depletion of the major gamma-tubulin isotype by RNA-mediated interference was assessed in the Drosophila S2 cell line. Depletion of gamma-tubulin has no significant effect on the cytoskeletal microtubules during interphase. However, it promotes an increase in the mitotic index, resulting mainly in monopolar and, to a lesser extent, asymmetrical bipolar prometaphases lacking astral microtubules. This mitotic accumulation coincides with the activation of the mitotic checkpoint. Immunostaining with an anti-Abnormal spindle (Asp) antibody revealed that the spindle poles, which were always devoid of gamma-tubulin, were unfocused and organized into sub-spindles. Despite the marked depletion of gamma-tubulin, the pericentriolar proteins CP190 and centrosomin were recruited to the spindle pole(s), where they formed three or four dots, suggesting the presence of several centrioles. Electron microscopic reconstructions demonstrated that most of the monopolar spindles exhibited three or four centrioles, indicating centriole duplication with a failure in the separation process. Most of the centrioles were shortened, suggesting a role for gamma-tubulin in centriole morphogenesis. Moreover, in contrast to metaphases observed in control cells, in which the spindle microtubules radiate from the pericentriolar material, in gamma-tubulin-depleted cells, microtubule assembly still occurs at the poles but involves the elongation of centriolar microtubule triplets. These results demonstrate that, after depletion of gamma-tubulin, the pericentriolar material is unable to promote efficient microtubule nucleation. They point to an alternative mechanism of centrosomal microtubule assembly that contributes to the formation of abnormal, albeit partially functional, mitotic spindles (Raynaud-Messina, 2004).
This study used RNAi to deplete the major gamma-tubulin isotype significantly in cultured Drosophila S2 cells. Only a few cells escaped this treatment, because less than 0.7% of all mitotic cells presented a bipolar morphology with a positive 23C gamma-tubulin signal at both poles. Depletion of 23C gamma-tubulin caused S2 cells to accumulate in mitosis, with an active mitotic checkpoint. They exhibited abnormal prometaphase stages, characterized by unseparated condensed chromatids and spindle poles with supernumerary short centrioles. The emergence of a characteristic phenotype (i.e. monopolar prometaphase figures) prompted an investigation of the mechanism of spindle organization in 23C-gamma-tubulin-depleted cells. This investigation revealed that, under gamma-tubulin-deficient conditions, the pericentriolar material is unable to efficiently promote microtubule nucleation. These observations point to the involvement of a previously undescribed centrosomal mechanism of microtubule assembly that operates via elongation of the distal centriolar extremities (Raynaud-Messina, 2004).
After depletion of 23C gamma-tubulin, the duplicated centrioles were present at the one pole of the monopolar prometaphase figures and at only one of the two poles in at least half of the bipolar figures, indicating a segregation failure. This phenomenon has also been suggested to occur in Drosophila Dgrip91 mutants. Observations made on mammalian cells have demonstrated that the relative positions of the two centriole pairs depend partly on the dynamics of the microtubule cytoskeleton. Hence, after gamma-tubulin depletion, it is likely that the microtubule subnetwork involved in the separation of the centriole pairs undergoes modifications in either its dynamics or in its density. Despite the lack of ultrastructural studies excluding morphogenetic and/or separation defects, basal-body duplication has been reported to be impaired after silencing of the gamma-tubulin-encoding genes in Paramecium. This discrepancy with the observations could result from differences in the timing of centriole and basal-body duplication, and the many rounds of basal-body duplication in Paramecium leading to many basal bodies. Thus, it is possible that the critical concentration of gamma-tubulin required for centriole duplication is lower than the one necessary for microtubule assembly by the pericentriolar material. In this regard, it is possible that the small amount of the 37CD gamma-tubulin isotype constitutively associated with the centrosomal fraction plays a role in centriole duplication. However, it is unlikely that this low-abundance gamma-tubulin isotype plays an important role in the nucleation of mitotic microtubules, as suggested by the absence of microtubules issuing directly from the pericentriolar material. After 23C gamma-tubulin depletion, the 37CD isotype was not affected; its expression level was maintained and it retained its polar localization. Moreover, under the experimental conditions used, the function of this minor gamma-tubulin isotype could not be determined by RNAi, despite the use of two distinct dsRNAs. The poor susceptibility of the 37CD gamma-tubulin isoform to the RNAi strategy might stem from its specific properties in Drosophila cells, such as its low concentration, its absence from the cytosolic fraction and/or a possible slow turnover (Raynaud-Messina, 2004).
In 23C gamma-tubulin-depleted S2 cells, the centrioles were often shorter than in the control cells. The presence of a short centriole among normal sized centrioles has been noticed in abnormal metaphases of neuroblasts from the Drosophila Dgrip91 mutants. Centrioles are generally believed to be very stable organelles. However, in Physarum amoebae, a transient decrease in the length of the parental centrioles occurs in mitosis, during the assembly of the daughter centrioles. If the gamma-tubulin present in the centriole and basal-body lumen interacts with the centriolar microtubules, its depletion might well alter centriole morphogenesis, possibly by an action on centriolar microtubule dynamics. Indeed, a role of gamma-tubulin in microtubule dynamics and stability has been suggested in Aspergillus, Caenorhabditis, as well as in mammalian cells, where gamma-tubulin interacts with the stable kinetochore microtubules. gamma-Tubulin-depleted Drosophila S2 cells mostly assembled half-spindles, resulting in monopolar metaphases with a normal overall microtubule orientation and a 13-protofilament structure. The presence of pericentriolar material was confirmed by electron microscopy and by the recruitment of the centrosomal proteins CP190 and centrosomin, which appeared to be independent of the presence of gamma-tubulin. Microtubule reassembly after cold treatment suggested that the pole retained a nucleation activity. However, consistent with the severe depletion of the main centrosomal gamma-tubulin, most of the spindle microtubules were not assembled from the pericentriolar material, which accounted for the absence of astral microtubules. Nucleation resulted at least partially from elongation of the distal extremity of the microtubules of the four centrioles. This process differs from the formation of primary cilia observed in several interphase animal cells. In mitotic 23C-gamma-tubulin-depleted S2 cells, the microtubules produced by the centrioles fail to form the characteristic axonemal structure. They are not surrounded by a membrane and are not associated in microtubule doublets as in typical primary cilia but rather diverge in the cytoplasm, giving rise to a few sub-spindles. Moreover, they did not arise from the dispersion of the ensheathing membrane of a primary cilium, as has been reported in cells undergoing early mitosis, because primary cilia were not observed in either control or treated interphase cells. It is likely that the distal extremity of the centriolar microtubules is not blocked by regulatory proteins and can assemble alpha-tubulin heterodimers, as observed in vitro with isolated centrioles. Unless triggered by the specific axonemal program, during normal mitosis, this elongation process remains exceptional. Observations suggest that this atypical elongation pathway is normally overridden by the efficiency of nucleation from the high number of gamma-tubulin nucleation sites present in the pericentriolar material. The failure of centriole separation and elongation of the centriolar microtubules that was observed is consistent with the formation of monopolar spindles. However, this assembly pathway might only give rise to a limited number of spindle microtubules, unless microtubules detach from the distal ends of the centriole. Alternatively, the presence of additional microtubule bundles on the chromosome face opposite the main pole suggests that microtubules might also be nucleated at the kinetochores. In contrast to the microtubule assembly pathway predominantly mediated by the pericentriolar gamma-tubulin, the results suggest that, below a critical concentration of gamma-tubulin, other assembly mechanisms could contribute to spindle formation. These alternative mechanisms are unable to generate a fully functional mitotic apparatus in most cases (Raynaud-Messina, 2004).
Taken together, the results demonstrate the inefficiency of the pericentriolar material in promoting microtubule nucleation after depletion of gamma-tubulin. They point to an alternative centrosome-driven mechanism of microtubule assembly that might contribute to the formation of abnormal, albeit partially functional, mitotic spindles (Raynaud-Messina, 2004).
Avides, M. C. O and Glover, D. M. (1999). Abnormal spindle protein, Asp, and the integrity of mitotic centrosomal microtubule
organizing centers. Science 283: 1733-1735
Barbosa, V., Yamamoto, R. R., Henderson, D. S. and Glover, D. (2000). Mutation
of a Drosophila gamma tubulin ring complex subunit encoded by discs degenerate-4
differentially disrupts centrosomal protein localization. Genes Dev. 14:
3126-3139. 11124805
Barbosa, V., et al. (2003). Drosophila dd4 mutants reveal that gammaTuRC is required to maintain juxtaposed half spindles in spermatocytes. J. Cell Sci. 116: 929-941. 12571290
Berdnik, D. and Knoblich, J. (2002). Drosophila Aurora-A is required for centrosome maturation and actin-dependent asymmetric protein localization during mitosis. Curr. Biol. 12: 640-647. 11967150
Casenghi, M., et al. (2003). Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation. Dev. Cell 5: 113-125. 12852856
Chang, P. and Stearns, T. (2000). Delta-tubulin and epsilon-tubulin: two new human centrosomal tubulins reveal new
aspects of centrosome structure and function. Nat. Cell Biol. 2(1): 30-35.
Colombi, N., et al. (2006). The Drosophila gamma-Tubulin small complex subunit Dgrip84 is required for structural and functional integrity of the
spindle apparatus. Molec. Biol. Cell 17: 272-282. 16236791
Combelles, C. M. H. and Albertini, D. F. (2001). Microtubule patterning during meiotic maturation in mouse oocytes is determined by cell cycle-specific sorting and redistribution of gamma-Tubulin. Dev. Bio. 239: 281-294
Debec, A., et al. (1995). Polar organization of gamma-tubulin in acentriolar mitotic spindles of Drosophila
melanogaster cells. J Cell Sci 108: 2645-2653.
Delgehyr, N., Sillibourne, J. and Bornens, M. (2005). Microtubule nucleation and anchoring at the centrosome are independent processes linked by ninein function. J. Cell Sci. 118(Pt 8): 1565-75. 15784680
Dictenberg, J. B., et al. (1998). Pericentrin and gamma-tubulin form a protein complex and are
organized into a novel lattice at the centrosome. J. Cell Biol. 141(1): 163-174.
Endow, S. A. and Komma, D. J. (1998). Assembly and dynamics of an anastral:astral spindle: the meiosis II spindle of Drosophila oocytes. J. Cell Sci. 111: 2487-2495.
Ferree, P. M., et al. (2006). The origin of centrosomes in parthenogenetic hymenopteran insects. Curr. Biol. 16: 801-807. 16631588
Geissler, S., Pereira, G., Spang, A., Knop, M., Soues, S., Kilmartin, J. and Schiebel, E. (1996). The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. EMBO J. 15: 3899-3911.
Goshima, G., et al. (2008). Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle. J. Cell Biol. 181: 421-429. PubMed Citation: 18443220
Grieder, N. C., de Cuevas, M. and Spradling, A. C. (2000). The fusome organizes the microtubule network during oocyte differentiation
in Drosophila. Development 127: 4253-4264
Gunawardane, R. N., et al. (2000). Characterization and reconstitution of Drosophila gamma-Tubulin ring complex subunits. J. Cell Biol. 151(7): 1513-1524. 11134079
Gunawardane, R. N., Martin, O. C. and Zheng, Y. (2003). Characterization of a new gammaTuRC subunit with WD repeats. Mol. Biol. Cell 14(3): 1017-26. 12631720
Helps, N., et al. (1998). Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos. J. Cell Sci. 111(10): 1331-1340
Hendrickson, T. W., et al. (2001). Conditional mutations in gamma-tubulin reveal its involvement in chromosome segregation and cytokinesis. Mol. Biol. Cell 12: 2469-2481. 11514629
Horio, T., et al. (1991). The fission yeast gamma-tubulin is essential for mitosis and is localized
at microtubule organizing centers.
J. Cell Sci. 99 ( Pt 4): 693-700
Horio, T. and Oakley, B. R. (1994). Human gamma-tubulin functions in fission yeast. J. Cell Biol. 126: 1465-1473
Januschke, J., et al. (2006). The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte. Development 133: 129-139. 16319114
Jaspersen, S. L., and Winey, M. (2004). The budding yeast spindle pole body: structure, duplication, and function. Annu. Rev. Cell Dev. Biol. 20: 1-28. 15473833
Joshi, H. C., et al. (1992).
Gamma-tubulin is a centrosomal protein required for cell
cycle-dependent microtubule nucleation. Nature 356: 80-3
Joshi, H. C. (1993). Gamma-tubulin: the hub of cellular microtubule assemblies. Bioessays 15: 637-43
Julian, M., et al. (1993). gamma-Tubulin participates in the formation of the midbody during
cytokinesis in mammalian cells. J. Cell Sci. 105 ( Pt 1): 145-56
Kawaguchi, S. and Zheng, Y. (2004). Characterization of a Drosophila centrosome
protein CP309 that shares homology with Kendrin and CG-NAP. Mol. Biol. Cell 15:
37-45. 14565985
Kellogg, D. R., et al. (1995). CP60: a microtubule-associated protein that is localized to the
centrosome in a cell cycle-specific manner. Mol. Biol. Cell 6: 1673-1684.
Kleylein-Sohn, J., Westendorf, J., Le Clech, M., Habedanck, R., Stierhof, Y. D. and Nigg, E. A. (2007). Plk4-induced centriole biogenesis in human cells.
Dev. Cell 13(2): 190-202. PubMed citation: 17681131
Kloc, M. and Etkin, L. D. (1998). Apparent continuity between the messenger transport
organizer and late RNA localization pathways during
oogenesis in Xenopus. Mech. Dev. 73(1): 95-106.
Knop, M., Pereira, G., Geissler, S., Grein, K. and Schiebel, E. (1997a). The spindle pole body component Spc97p interacts with the gamma-tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication. EMBO J. 16: 1550-1564.
Knop, M. and Schiebel, E. (1997b). Spc98p and Spc97p of the yeast gamma-tubulin complex
mediate binding to the spindle pole body via their
interaction with Spc110p. EMBO J. 16(23): 6985-6995.
Knop, M. and Schiebel, E. (1998). Receptors determine the cellular localization of a gamma-tubulin
complex and thereby the site of microtubule formation. EMBO J. 17(14): 3952-3967.
Lange, B. M. and Gull, K. (1995). A molecular marker for centriole maturation in the mammalian cell cycle. J. Cell Biol. 130(4): 919-27.
Lee, C., Scherr, H. M. and Wallingford, J. B. (2007). Shroom family proteins regulate gamma-tubulin distribution and microtubule architecture during epithelial cell shape change. Development 134: 1431-1441. Medline abstract: 17329357
Llamazares, S., Tavosanis, G. and Gonzalez, C. (1999). Cytological characterisation of the mutant phenotypes produced during early embryogenesis by null and loss-of-function alleles of the gammaTub37C gene in Drosophila. J. Cell Sci. 112 (Pt 5): 659-67.
Luders, J., Patel, U. K. and Stearns, T. (2006). GCP-WD is a gamma-tubulin targeting factor required for centrosomal and chromatin-mediated microtubule nucleation. Nat. Cell Biol. 8(2): 137-47. 16378099
Mahoney, N. M., Goshima, G., Douglass, A. D. and Vale, R. D. (2006). Making microtubules and mitotic spindles in cells without functional centrosomes.
Curr. Biol. 16(6): 564-9. 16546079
Marschall, L. G., et al. (1996). Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for
microtubule-organizing center function. J. Cell Biol. 134: 443-454.
Martin, O. C., et al. (1998). Xgrip109: A gamma Tubulin-associated protein with an essential role in gamma tubulin ring complex (gammaTuRC) assembly and centrosome function. J. Cell Biol. 141(3): 675-687.
McKean, P. G., et al. (2003). gamma-Tubulin functions in the nucleation of a discrete subset of microtubules in the eukaryotic flagellum. Curr. Biol. 13: 598-602. 12676092
Moritz, M., Braunfeld, M. B., Fung, J. C., Sedat, J. W., Alberts, B. M. and Agard,
D. A. (1995a). Three-dimensional structural characterization of centrosomes from early Drosophila embryos. J. Cell Biol. 130: 1149-1159.
Moritz, M., et al. (1995b). Microtubule nucleation by gamma-tubulin-containing rings in the
centrosome. Nature 378: 638-64
Moritz, M., et al. (2000). Structure of the gamma-tubulin ring complex: a template for microtubule nucleation. Nat. Cell Biol. 2: 365-370.
Muller, E. G., et al. (2005). The organization of the core proteins of the yeast spindle pole body. Mol. Biol. Cell 16(7): 3341-52. 15872084
Murphy, S. M., Urbani, L. and Stearns, T. (1998). The mammalian gamma-Tubulin complex contains homologues of the yeast spindle pole body components Spc97p and Spc98p. J. Cell Biol. 141(3): 663-674.
Nakamura, M., et al. (1998). When overexpressed, a novel centrosomal protein, RanBPM, causes
ectopic microtubule nucleation similar to gamma-tubulin. J. Cell Biol. 143(4): 1041-52.
Oakley, B. R., et al. (1990). Gamma-tubulin is a component of the spindle pole body that is essential
for microtubule function in Aspergillus nidulans.
Cell 61: 1289-301
Oegema, K., Whitfield, W. G. F., and Alberts, B. (1995). The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains. J. Cell Biol. 131: 1261-1273
Oegema, K., et al. (1997). Two proteins that cycle asynchronously between
centrosomes and nuclear structures: Drosophila CP60
and CP190. J. Cell Sci. 110( Pt 14): 1573-1583.
Oegema, K., et al. (1999). Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules. J. Cell Biol. 144(4): 721-733. 10037793
Pereira, G., et al. (1999). Interaction of the yeast gamma-tubulin complex-binding protein
Spc72p with Kar1p is essential for microtubule function during
karyogamy. EMBO J. 18: 4180-4195.
Paluh, J. L., et al. (2000). A mutation in gamma-Tubulin alters microtubule dynamics and organization and is synthetically lethal with the Kinesin-like protein
Pkl1p. Mol. Biol. Cell 11: 1225-1239.
Raff, J. W., Kellogg, D. R. and Alberts, B. M. (1993).
Drosophila gamma-tubulin is part of a complex containing two
previously identified centrosomal MAPs. J. Cell Biol. 121: 823-35.
Raynaud-Messina, B., Debec, A., Tollon, Y., Gares, M. and Wright, M. (2001).
Differential properties of the two Drosophila gamma-tubulin isotypes. Eur. J. Cell
Biol. 80: 643-649. 11713868
Raynaud-Messina, B., Mazzolini, L., Moisand, A., Cirinesi, A. M. and Wright, M.
(2004). Elongation of centriolar microtubule triplets contributes to the
formation of the mitotic spindle in gamma-tubulin-depleted cells. J. Cell Sci. 117:
5497-5507. 15479719
Riparbelli, M. G. and Callaini, G. (1996). Meiotic spindle organizatin in fertilized Drosophila oocyte: presence of centrosomal components in the meiotic apparatus. J. Cell Sci. 109: 911-918.
Riparbelli, M. G., et al (1997). Assembly of the zygotic centrosome in the fertilized Drosophila egg. Mech. Dev. 65(1-2): 135-144.
Rosales-Nieves, A. E., et al. (2006). Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino.
Nat. Cell Biol. 8(4): 367-76. 16518391
Ruiz, F., et al. (1999). Basal body duplication in Paramecium requires gamma-tubulin. Curr. Biol. 9(1): 43-46
Sage, C. R., et al. (1995). Site-directed mutagenesis of putative GTP-binding sites of yeast
beta-tubulin: evidence that alpha-, beta-, and gamma-tubulins are atypical GTPases.
Biochemistry 34: 7409-7419
Salas, P. J. I. (1999). Insoluble gamma-Tubulin containing structures are anchored to
the apical network of intermediate filaments in polarized CACO-2
epithelial cells. J. Cell Biol. 146: 645-658.
Sampaio, P., et al. (2001). Organized microtubule arrays in gamma-tubulin-depleted Drosophila spermatocytes. Cur. Bio. 11: 1788-1793. 11719222
Shu, H. B. and Joshi, H. C. (1995). Gamma-tubulin can both nucleate microtubule assembly and
self-assemble into novel tubular structures in mammalian cells. J. Cell Biol. 130: 1137-1147
Sibon, O. C. M., et al. (2000). DNA-replication/DNA-damage-dependent centrosome inactivation in Drosophila embryos. Nat. Cell Biol. 2: 90-95.
Sobel, S. G. and Synder, M. (1995). A highly divergent gamma-tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae. J. Cell Biol. 131: 1775-88
Spang, A., et al. (1996).
gamma-Tubulin-like Tub4p of Saccharomyces cerevisiae is associated
with the spindle pole body substructures that organize microtubules and
is required for mitotic spindle formation. J. Cell Biol. 134: 429-441
Strome, S., et al. (2001). Spindle dynamics and the role of gamma-tubulin in early Caenorhabditis elegans embryos. Mol. Biol. Cell 12: 1751-1764. 11408582
Stumpff, J., et al. (2005). Drosophila Wee1 interacts with members of the γTURC and is required for proper mitotic-spindle morphogenesis and positioning. Curr. Biol. 15: 1525-1534. 16139207
Sunkel, C. E., et al. (1995). Gamma-tubulin is required for the structure and function of the
microtubule organizing centre in Drosophila neuroblasts. EMBO J. 14: 28-36
Takahashi, M., Yamagiwa, A., Nishimura, T., Mukai, H. and Ono, Y. (2002).
Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by
anchoring gamma-tubulin ring complex. Mol. Biol. Cell 13: 3235-3245. 12221128
Tassin, A. M., et al. (1998). Characterization of the human homologue of the yeast Spc98p and its association with gamma-Tubulin. J. Cell Biol. 141(3): 689-701.
Tavosanis, G., et al. (1997). Essential role for gamma-tubulin in the acentrolar female meiotic spindle of
Drosophila. EMBO J. 16: 1809-19.
Terada, Y., Uetake, Y. and Kuriyama, R. (2003). Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells.
J. Cell Biol. 162(5): 757-63. 12939255
Vardy, L. and Toda, T. (2000). The fission yeast gamma-tubulin complex is required in G1 phase and is a component of the spindle assembly checkpoint. EMBO J. 19: 6098-6111.
Vardy, L., Fujita, A. and Toda, T. (2002). The gamma-tubulin complex protein Alp4 provides a link between the metaphase checkpoint and cytokinesis in fission yeast. Genes Cells 7: 365-373. 11952833
Verollet, C., Colombie, N., Daubon, T., Bourbon, H. M., Wright, M. and Raynaud-Messina, B. (2006). Drosophila melanogaster γTuRC is dispensable for targeting γ-tubulin to the centrosome and microtubule nucleation. J. Cell Biol. 172: 517-528. Medline abstract: 16476773
Vogel, J., et al. (2001). Phosphorylation of gamma-tubulin regulates microtubule organization in budding yeast. Dev. Cell 1: 621-631. 11709183
Vogt, N., Koch, I., Schwarz, H., Schnorrer, F. and Nusslein-Volhard, C. (2006).
The gammaTuRC components Grip75 and Grip128 have an essential microtubule-anchoring function in the Drosophila germline. Development 133(20): 3963-72. Medline abstract: 16971473
Wang, Y. and Riechmann, V. (2008). Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm. Mech. Dev. 125(1-2): 142-52. PubMed citation: 18053693
Wilson, P. G., et al. (1997). Differential expression of two gamma-Tubulin isoforms during gametogenesis and development in Drosophila. Dev. Biol. 184: 207-221.
Whitfield, W. G., et al. (1995). The 190 kDa centrosome-associated protein of Drosophila
melanogaster contains four zinc finger motifs and binds to specific
sites on polytene chromosomes. J. Cell Sci. 108: 3377-3387.
Young, A., et al. (2000). Cytoplasmic dynein-mediated assembly of pericentrin and
gammaTubulin onto centrosomes. Mol. Biol. Cell 11: 2047-2056.
Zhang, L., et al. (2000). The role of Xgrip210 in gamma-Tubulin ring complex assembly and centrosome recruitment. J. Cell Biol. 151(7): 1525-1536. 11134080
Zhang, J. and Megraw, T. L. (2007). Proper recruitment of gamma-tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires Centrosomin Motif 1.
Mol. Biol. Cell 18(10): 4037-49. Medline abstract: 17671162
Zheng, Y., Jung, K. and Oakley, B. R. (1991). Gamma-Tubulin is present in Drosophila melanogaster and Homo sapiens and is associated with centrosomes. Cell 65: 817-23
Zheng, Y., et al. (1995). Nucleation of microtubule assembly by a gamma-tubulin-containing ring
complex. Nature 378: 578-583
Zimmerman, W. C., Sillibourne, J., Rosa, J. and Doxsey, S. J. (2004).
Mitosis-specific anchoring of gamma-tubulin complexes by pericentrin controls spindle organization and mitotic entry. Mol. Biol. Cell 15: 3642-3657. 15146056
gammaTubulin at 23C:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 20 August 2008
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