gammaTubulin at 23C
In metazoans, γ-tubulin acts within two main complexes, γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs). In higher eukaryotes, it is assumed that microtubule nucleation at the centrosome depends on γ-TuRCs, but the role of γ-TuRC components remains undefined.
This study analyzed the function of all four γ-TuRC–specific subunits in Drosophila: Dgrip75, Dgrip128, Dgrip163, and Dgp71WD. Grip-motif proteins, but not Dgp71WD, appear to be required for γ-TuRC assembly. Individual depletion of γ-TuRC components, in cultured cells and in vivo, induces mitotic delay and abnormal spindles. Surprisingly, γ-TuSCs are recruited to the centrosomes. These defects are less severe than those resulting from the inhibition of γ-TuSC components and do not appear critical for viability. Simultaneous cosilencing of all γ-TuRC proteins leads to stronger phenotypes and partial recruitment of γ-TuSC. In conclusion, γ-TuRCs are required for assembly of fully functional spindles, but it is suggested that γ-TuSC could be targeted to the centrosomes, which is where basic microtubule assembly activities are maintained (Verollet, 2006; full text of article).
Individual depletion of Dgrip75, Dgrip128, or Dgrip163 induced a strong decrease in cytoplasmic γ-TuRCs in cultured S2 cells. This is consistent with previous data obtained in vitro showing that immunodepletion of Xgrip210, the Xenopus laevis orthologue of Dgrip163, blocks the reassembly of purified and salt-treated γ-TuRCs. In contrast to the strong decrease of γ-TuRCs observed after the depletion of grip-motif proteins, the down-regulation of Dgp71WD did not produce a significant effect on the ratio and the sedimentation coefficients of the γ-tubulin complexes. However, the possibility that residual amounts of this protein are sufficient to maintain the overall structure of these complexes cannot be excluded. Dgp71WD is probably associated with the γ- TuRCs at a low stoichiometry, explaining why no change in the sedimentation coefficient could be detected after its depletion. The data suggest that although the grip-motif proteins are essential for the assembly and stability of γ-TuRCs, Dgp71WD appears dispensable for these processes (Verollet, 2006).
In Dgp71WD-depleted cells, no obvious effect was observed on the level of cytosolic γ-TuRCs and on the recruitment of the different γ-TuRC components to the mitotic centrosomes, suggesting that γ-TuRCs depleted of Dgp71WD had not completely lost their ability to be targeted to the poles. However, γ-tubulin localization along spindle microtubules was impaired and monopolar figures with poorly separated poles appeared with a high occurrence. These monopolar phenotypes are similar to those observed after depletion of Nedd1, the human Dgp71WD orthologue. The mitotic defects could result from a partial functionality of the γ-tubulin complexes recruited to the centrosomes or from modification in the properties of microtubule arrays that were no longer decorated by γ-tubulin. Thus, it appears that the complete γ-TuRC is required for normal mitotic progression (Verollet, 2006).
If one assumes that γ-tubulin is recruited to the centrosome in the form of γ-TuRCs, depletion of γ-TuRCs and γ-tubulin would be expected to lead to similar phenotypes. In fact, depletion of grip-motif components of the γ-TuRCs, such as Dgrip75, did not fully mimic the effects of depletion of γ-TuSC subunits on mitotic progress. Less severe defects were observed as indicated by the lower occurrence of monopolar spindles, the efficient distribution of centrosomes to the two poles, the maintenance of bipolar spindles with astral microtubules and focused poles. These phenotypes had been confirmed in vivo, using mutant larval neuroblasts. These moderate defects were consistent with the viability of Dgrip75 mutants and the weak percentage of aneuploidy. Surprisingly, despite a significant reduction of the cytosolic pool of γ-TuRCs, the tethering of the three γ-TuSC proteins to the centrosomes was not impaired, as judged by immunofluorescence analyses both in mutant neuroblasts and in cultured cells. Similar phenotypes were observed after individual silencing of the two other grip-motif proteins, Dgrip128 and Dgrip163. It was noteworthy that after an RNAi treatment against any one of the three grip-motif proteins specific of the γ-TuRCs, a decrease in the level of the two others was noticed, suggesting some coregulation between this set of proteins. Altogether, these results show that the complete γ-TuRC is not a prerequisite for the centrosomal accumulation of γ-TuSC proteins (Verollet, 2006).
Because γ-tubulin could be recruited to the centrosomes independently of the γ-TuRCs, characterization of the complex that mediates its targeting was of interest. Sucrose gradient and immunofluorescence analyses after depletion of individual γ-TuRC components strongly suggest that γ-tubulin is recruited to the centrosomes as γ-TuSC. Codepletion of the four γ-TuRC–specific proteins still allowed the polar accumulation of the γ-TuSC components in most of the cells. This latter experiment supports the hypothesis that the γ-TuSC is a vector competent for targeting γ-tubulin to the centrosome. However, since RNAi treatments do not completely remove all protein, it is possible that the residual γ-TuRC–specific proteins participate in the formation of a γ-TuRC–like structure that is unstable in the cytoplasm, but stabilized upon assembly within the pericentriolar matrix. The idea that γ-TuSC could be recruited to the poles is consistent with data reported in Saccharomyces cerevisiae, in which no orthologues of the γ-TuRC–specific proteins have been reported. In this organism, the main interactions of γ-tubulin with the microtubule-organizing centers are mediated by the two γ-tubulin–associated proteins in the γ-TuSC. Similarly, in mammals, the γ-TuRCs can be tethered to the centrosomes via interactions of the orthologues of Dgrip84 and Dgrip91 with the centrosomal anchoring proteins kendrin and centrosome- and Golgi-localized protein kinase N–associated protein. In Drosophila, the calmodulin-binding protein CP309 has been proposed to anchor γ-TuRCs to the centrosome by direct binding to the γ-TuSC. However, γ-tubulin recruitment to the centrosomes is probably a complex process, as additional proteins, including ninein and centrosomin, provide alternative sites for γ-tubulin anchorage. Although evidence suggests that γ-tubulin can be targeted to the centrosomes in the form of γ-TuSCs, it cannot be ruled out that in Drosophila other proteins at low abundance, previously uncharacterized proteins such as the Dgrip79 and Dgrip223 identified by in silico analyses, or different combinations of known γ-tubulin partners could be involved in this recruitment. Moreover, after concomitant depletion of all γ-TuRC–specific proteins, spindles were nonfunctional and the amount of γ-TuSC recruited to the mitotic centrosomes was reduced. Because of the observation that the small complexes are less active than γ-TuRCs in promoting nucleation, it is proposed that the decrease in the amount of γ-TuSCs under a threshold can be critical for spindle functionality (Verollet, 2006).
Interestingly, the transient association of γ-tubulin to the spindle, as well as to the midbody, was no longer detectable after individual or concomitant RNAi treatment against Dgrip75, Dgrip128, Dgrip163, and/or Dgp71WD. Hence, mitotic γ-tubulin localization to structures outside the pericentriolar material requires the fully assembled γ-TuRCs, and Dgp71WD could play an active role in this process. The absence of γ-tubulin recruitment along the spindle microtubules and at the midbody can also be explained by distinct properties of docking proteins at centrosomes and at noncentrosomal sites. Actually, novel proteins in charge of the recruitment of γ-tubulin complexes along spindle microtubules have recently been identified in fission yeast (Verollet, 2006).
In contrast with the impairment of microtubule assembly from the pericentriolar material after removal of γ-TuSC components, the pericentriolar material appears to remain active as a microtubule-nucleating center after down-regulation of specific γ-TuRC proteins, as shown by the presence of astral microtubules identified both by immunofluorescence and electron microscopy. Moreover, microtubules containing 13 protofilaments were still nucleated, challenging the 'template model'. This indicates that preassembly of cytosolic γ-TuRCs does not seem required for the formation of canonical microtubules. It is not excluded that a ringlike structure could be assembled from γ-TuSCs alone or in combination with small amounts of γ-TuRC–specific proteins at the pericentriolar level, although the purified γ-TuSC does not assemble into larger structures in vitro. In that case, such ringlike complexes would exhibit stoichiometries and protein compositions different from γ-TuRCs (Verollet, 2006).
These observations could reconcile the findings of microtubule nucleation in animal cells and in S. cerevisiae. Moreover, after depletion of γ-TuRC–specific components, recruitment of γ-TuSC appeared sufficient, in most of the cases, to control the formation of spindles competent for chromosome segregation. However, mitotic processes were partly disrupted in cells lacking γ-TuRCs, leading to a transient prometaphase accumulation and a poor density of spindle microtubules. Several possibilities could account for these defects, such as a lower efficiency of γ-TuSCs compared with γ-TuRCs in microtubule nucleation, the presence of distinct binding sites for γ-TuSCs and γ-TuRCs, or the loss of γ-TuRC recruitment along spindle microtubules, which would affect the organization or the dynamics of specific microtubule arrays (Verollet, 2006).
Collectively, the results strongly suggest that the assembly of γ-TuRCs is not essential for γ-tubulin–dependent microtubule nucleation at the centrosome, but instead is required for other, noncentrosomal localization of γ-tubulin. In Drosophila, γ-TuRCs may target a fully organized 'nucleation machinery' to the sites of nucleation; γ-TuSC would act as a functional unit, whereas γ-TuRC–specific proteins would rather play a more refined role in regulating or optimizing of microtubule arrays during mitosis. γ-Tubulin can be targeted to the poles by the direct docking of γ-TuSCs that exert basic nucleation activity. This γ-TuSC recruitment could be a 'salvage pathway,' involved only when the dominant microtubule assembly mechanism mediated by γ-TuRCs is impaired or as a physiological pathway acting in parallel to γ-TuRC nucleation activity. Altogether, the data should prompt the reexamination of the current nucleation models (Verollet, 2006).
The γ-tubulin ring complex (γTuRC) forms an essential template
for microtubule nucleation in animal cells. The molecular composition of the
γTuRC has been described; however, the functions of the subunits
proposed to form the cap structure remain to be characterized in vivo. In
Drosophila, the core components of the γTuRC are essential for
mitosis, whereas the cap component Grip75 is not required for viability but
functions in bicoid RNA localization during oogenesis. The other cap
components have not been analyzed in vivo. This study reports the functional
characterization of the cap components Grip128 and Grip75. Animals with
mutations in Dgrip128 or Dgrip75 are viable, but both males
and females are sterile. Both proteins are required for the formation of
distinct sets of microtubules, which facilitate bicoid RNA
localization during oogenesis, the formation of the central microtubule aster
connecting the meiosis II spindles in oocytes and cytokinesis in male meiosis.
Grip75 and Grip128 anchor the axoneme at the nucleus during sperm elongation.
It is proposed that Grip75 and Grip128 are required to tether microtubules at
specific microtubule-organizing centers, instead of being required for general
microtubule nucleation. The γTuRC cap structure may be essential only
for non-centrosome-based microtubule functions (Vogt, 2006).
γTuRC function in microtubule nucleation is crucial for viability;
mutations in Grip91/l(1)dd4, Grip84 and γTub23C are
lethal. By contrast, Grip75, Grip128 and the double mutants are viable, showing that both gene products are not essential for the microtubule-nucleating
properties of the γTuRC and that the γTuRC formed in these mutants
is sufficient for microtubule function in somatic cell types of the fly.
However, depletion of cap components such as Grip75, Grip128 or Grip163 by
RNAi leads to a higher mitotic index in S2 cells, but
the cap components are not absolutely essential for mitotic progression. This
is not surprising as even mutants with centrosomal defects can survive. Furthermore, γ-tubulin is recruited to centrosomes in
Grip75 or Grip128 mutant spermatocytes, Grip75
mutant neuroblasts and in S2 cells depleted for cap components, showing that γ-tubulin targeting to the centrosome
does not depend on cap components. It has been proposed that γ-tubulin
can be recruited to centrosomes as part of the γTuSC, as the amount of
large γ-tubulin-containing complexes is severely reduced in cells
depleted for cap components. Using buffers with lower salt concentrations, we
observe large γ-tubulin containing complexes in Grip75 and
Grip128 mutants, albeit in reduced amounts compared with wild type.
It is likely that these complexes are indeed γTuRCs that lack parts of
the cap structure, as they are similar in size to the γTuRC; in
addition, Grip128 is present in Grip75 mutant complexes. The mutant
γTuRCs might still be capable of nucleating microtubules (Vogt, 2006).
Whether γ-tubulin forms γTuSCs or incomplete γTuRCs, the
cap subunits are dispensable for microtubule nucleation and γ-tubulin
recruitment to centrosomes. Moreover, a γTuRC has not been described in Saccharomyces cerevisiae and homologs of the cap components have not been identified in yeast, further supporting the notion that microtubule nucleation can occur in the absence of the cap structure (Vogt, 2006).
In Drosophila, it is not known whether individual γTuRC
complexes vary in their subunit composition and whether the
γTuRC-specific subunits have similar functions. The human γTuRC
has been shown to contain all of the described subunits. The cap components Grip75, Grip128 and Grip163 depend on each other for their stability. Furthermore, individual depletion of Grip75, Grip128 or Grip163 results in a similar increase of the mitotic index in treated cells. Moreover, Grip128;Grip75 double mutants show the same phenotypes as the single mutants in the Drosophila germline. Taken together, the data support the view that Grip163, Grip128 and Grip75 function in the same processes and are part of the same complexes (Vogt, 2006).
By contrast, Grip71 appears to have a distinct function. On the one hand,
depletion by RNAi does not impair protein levels of the other
γTuRC-specific proteins or their recruitment to centrosomes; on the
other hand, the mitotic phenotypes are much stronger in Grip71
mutants when compared with Grip75 mutants (Vogt, 2006).
Genetic and cell biological data suggest that an intact cap structure is
not necessary for microtubule nucleation; thus,
the function of the cap is still in question. It could be required for
efficient assembly of the γTuRC, for a higher microtubule nucleation
rate or for tethering the complex to MTOCs. The former two possibilities
predict that all microtubules would be affected to a similar degree, and
therefore the most sensitive microtubule-dependent processes would be
disrupted in Grip75 and Grip128 mutants. The latter
possibility predicts that phenotypes would arise when redundant anchoring
mechanisms were not available (Vogt, 2006).
Mutants with global defects in microtubule function such as hypomorphic
αtub84B mutants show a wide range of phenotypes such as
polyphasic lethality, cuticle defects, short life span and sterility.
Similarly, hypomorphic Grip91/l(1)dd4 mutants are lethal and display
both mitotic and meiotic defects in spermatogenesis. As
Grip75 and Grip128 mutants show very specific phenotypes, a
function for the γTuRC cap structure in microtubule anchoring at MTOCs
is more conceivable. This is supported by the observed detachment of axonemes
from their respective nuclei without any aberrations in axoneme architecture
and the undisturbed orb RNA localization in Grip128 mutants.
Microtubule recruitment to or anchoring at centrosomes has been shown to
depend on a number of factors, such as pericentrin or motor proteins.
Redundant mechanisms might act to focus microtubules at conventional MTOCs in
somatic cells, but this might not be the case at nonconventional MTOCs in the
Drosophila germline (Vogt, 2006).
The Drosophila pericentrin-like protein D-PLP recruits or anchors
γ-tubulin to centrosomes, possibly by direct interaction with
γTuSC components. Interestingly, D-PLP is only required for efficient
anchoring of γ-tubulin to the centrosome in early phases of mitosis,
suggesting that a D-PLP independent pathway can recruit and anchor centrosomal
components. Maybe D-PLP and the γTuRC cap structure act redundantly in anchoring γ-tubulin at the pericentriolar material during mitosis (Vogt, 2006).
Additionally, microtubule motors focus microtubules at the mitotic
centrosome. Inhibition of the dynein-dynactin complex results in disorganized
spindles that lack well-focused poles, while
analysis of Dhc64C mutations in Drosophila suggests that
dynein is required for the attachment of spindle poles at centrosomes. The
kinesin-related Ncd is a minus-end directed microtubule motor that also
functions in spindle assembly during mitosis. Depletion
of Ncd by RNAi in S2 cells results in frequent release of microtubules from
the spindle pole (Vogt, 2006).
Although the roles of D-PLP and the above mentioned microtubule motors are
fairly well established in centrosomes, their contributions to other MTOCs,
such as the Grip75- and Grip128-dependent ones, are not as well studied. D-PLP
has been shown to maintain the structural integrity of centrioles in male
meiosis I; however, a possible function in γ-tubulin anchoring
in male meiosis II is difficult to address because of the centriolar defects.
Ncd organizes the female meiosis I spindle and
also localizes to the meiosis II spindle. ncd mutants do not form a
structured central aster in meiosis II, but
this could be a consequence of defects in meiosis I. It is proposed that redundant
mechanisms focus or anchor microtubules at conventional centrosomes during
mitosis. However, some MTOCs in the germline crucially depend on the anchoring
function of the γTuRC cap subunits Grip128 and Grip75. Hence, these
proteins allow the organization of distinct microtubule populations at
particular positions in complex cells, independently of centrosomes (Vogt, 2006).
Interestingly, mutations in Grip75 or Grip128 fully
disrupt the function of only certain MTOCs. As Grip128 and
Grip75 mutants are viable, most microtubule-dependent processes in
somatic cells function at least to an extent that allows survival of the
organism, even though mitosis is delayed. These
processes are directed by microtubules associated with classical centrosomes,
suggesting that somatic centrosomes are less sensitive to the lack of
Grip128 and Grip75 function than the specialized MTOCs in
the male and female germline (Vogt, 2006).
Grip128, Grip75 and γTub37C participate in the formation of a new
MTOC at stage 10b, which directs the relocalization of bcd RNA during
stage 10b. They are specifically involved in bcd RNA
localization; other microtubule-dependent processes in the oocyte such as
oocyte specification, nuclear migration, cytoplasmic streaming, and orb,
grk and osk RNA transport are normal in the respective mutants.
It has been proposed that different subsets of microtubules could perform this
variety of functions. Alternatively, loss of Grip128 or Grip75
function could lead to a reduction in microtubule number or function, thus
impairing only the most sensitive microtubule-dependent processes. Three lines
of evidence support a selective function of Grip128 in the organization of the
anteriorly originating microtubules during stage 10b and 11. The subcortical
microtubule network appears to be normal in mutant oocytes, whereas the
anterior set of microtubules is not present. Cytoplasmic streaming is
undisturbed in Grip128 mutants. orb RNA localization has
been demonstrated to be more sensitive to microtubule-depolymerizing drugs
than bcd RNA localization; however, orb RNA is correctly localized in Grip128 mutant oocytes. These data argue against a general
microtubule impairment in Grip128 mutants (Vogt, 2006).
Female meiosis requires the activities of Grip128 and Grip75 during the
second meiotic division. Spindle formation in female meiosis is atypical, with
the anastral and acentrosomal first meiotic spindle forming in a
chromatin-driven fashion. The second meiotic division is characterized by two
tandemly arranged spindles, which are connected by a central microtubule
aster. This central aster has been proposed to be necessary for correct
spacing and alignment of the meiosis II spindles. It contains γ-tubulin, whereas the distal poles are devoid of γ-tubulin. The absence of the central microtubule aster in Grip75 and Grip128 mutants could be due either to reduced microtubule nucleation from the MTOC or to a failure in MTOC assembly. The latter hypothesis is favored, since the inner half spindles are formed in the
mutants, and the absence of a robust central microtubule aster is also
observed in cnn and polo mutants (Vogt, 2006).
As in females, meiosis in males displays special features, such as the
reductional segregation of centrioles in the second meiotic division. Thus,
the second meiotic spindle is built from centrosomes, which contain a single
centriole each, thereby giving rise to unicentriolar cells.
Centrioles in spermatocytes are large and associated with very little
pericentriolar material when compared with mitotic centrioles.
These meiotic centrosomes might depend on Grip75 and Grip128 for correct
microtubule organization. Alternatively, the central spindle, which is
essential for cytokinesis, has been postulated to use transient microtubule
organizing centers present between the two daughter nuclei. Grip75 and Grip128
could function in these transient MTOCs to organize the central spindle (Vogt, 2006).
The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. This study reports a careful analysis of the oocyte cytoskeleton. Thick actin bundles are identified at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, the data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. Actin bundle formation requires Profilin but not Capu and Spire. Thus, these results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, these data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. The release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent (Wang, 2007).
This study reports the existence of actin bundles at the cortex of the oocyte which are involved in the cortical localisation of γTubulin. γTubulin is part of the γTubulin ring complex that is stabilising the minus ends of MTs. The presence of γTubulin alone does not allow distinguishing whether the protein is part of a microtubule organising centre (MTOC) that nucleates MTs or whether it only protects existing MTs from depolymerisation. Here, γTubulin was used solely as a maker for the MT minus ends, and it was shown that these are embedded within the cortical actin bundles before stage 10b (Wang, 2007).
The cytoskeletal rearrangements at stage 10b include the disassembly of the cortical actin bundles, the redistribution of the MT minus ends from the cortex to subcortical regions and the formation of MT arrays parallel to the oocyte cortex. Concomitantly with these cytoskeletal changes, the transition from slow to fast cytoplasmic streaming is triggered. What is the causal relationship between these events? The finding that interfering with actin bundle formation by drug treatment and GFPactin5c overexpression results in MT minus ends redistribution, MT array formation and premature fast streaming indicates that actin bundling acts upstream of MT reorganisation and streaming. The analysis of Khc mutants allows to further dissect the subsequent steps reorganising the MT cytoskeleton. In the absence of streaming, caused by the loss of Khc function, the redistribution of MT minus ends occurs normally, while the formation of MT arrays is abolished. Thus, minus end redistribution is upstream of streaming, and array formation is downstream. It is therefore proposed that streaming is initiated by the disassembly of the cortical actin bundles resulting in loss of cortical MT minus end anchoring. It is further proposed that the redistribution of the minus ends to subcortical regions is important for the reorganisation of the MT cytoskeleton into arrays that run parallel to the oocyte cortex. At this step a previously suggested self amplifying loop could be initiated, in which MT array formation and Kinesin movement enhance each other. In this loop the Kinesin driven streaming helps to sweep MTs into parallel arrays, which in turn allow more robust currents in the cytoplasm (Wang, 2007 and references therein).
How do the actin binding proteins Capu, Spire and Profilin act on the oocyte cytoskeleton to prevent premature cytoplasmic streaming? chic/Profilin mutants and latrunculin A treatment both interfere with bundle formation. Latrunculin A treatment inhibits actin polymerisation by binding to and sequestering actin monomers. Profilin is involved in actin polymerisation by delivering actin monomers to the growing ends of actin filaments. Thus, latrunculin A and Profilin mutants appear to interfere with bundling by limiting the pool of monomers that can be added to growing actin filaments. In contrast, capu and spire mutants are not required for the formation of actin bundles. It is proposed that Capu and Spire anchor the MT minus ends in a scaffold provided by the cortical actin bundles. The lack of Capu and Spire activity in the mutants prevents cortical MT anchoring and allows streaming in the presence of actin bundles. This model is supported by the work of Rosales-Nieves (2006) who have shown that Capu and Spire proteins are able to crosslink F-actin and MTs in vitro, and that both proteins localise to the oocyte cortex (Wang, 2007).
The regulation of fast ooplasmic streaming could be controlled at the level of the cortical localisation of Capu and Spire. The displacement of the two proteins from the cortex at stage 10b might result in loss of MT minus end anchoring, and thereby induce fast streaming. To test this, the localisation of GFP-Capu and GFP-Spire was analysed in cross sections of oocytes. However, no difference were in the localisation of the two proteins before and after onset of fast streaming. In addition, no displacement of GFP-Capu and GFP-Spire was detected after induction of premature streaming by latrunculin A treatment. Thus, Capu and Spire activities are not regulated at the level of their localisation (Wang, 2007).
A different mode of Capu and Spire regulation is suggested by their genetic and biochemical interaction with Rho1. This interaction led to a model in which Rho1 initiates fast streaming by regulating the crosslinking activities of Capu and Spire (Rosales-Nieves, 2006). The prevention of streaming requires not only capu and spire but also the presence of actin bundles. The formation of these bundles occurs, however, independently of capu and spire. This suggests that the onset of fast streaming is not only controlled by regulating Capu and Spire activities, but also by disassembly of the actin bundles (Wang, 2007).
Genes were also tested that are involved in actin regulation in the oocyte but do not induce premature streaming. For capulet, swallow and moesin mutants the formation of ectopic actin clumps has been reported reflecting defects in the organisation of the oocyte actin cytoskeleton. The presence was confirmed of ectopic F-actin in the oocyte cytoplasm in these mutants, but nevertheless the formation cortical actin bundles was detected. Thus, actin defects in the oocyte do not necessarily affect cortical actin bundling (Wang, 2007).
Since the discovery of γ-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of γ-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for γ-tubulin might reside elsewhere. An RNA interference (RNAi) screen, carried out in Drosophila cultured cells, has identified five genes (Dgt2-6) required for localizing γ-tubulin to spindle microtubules. The Dgt proteins interact, forming a stable complex. Spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex, named 'augmin' functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. A functional human homologue of Dgt6 has also been identified. These results suggest that an important mitotic function for γ-tubulin may lie within the spindle, where augmin and γ-tubulin function cooperatively to amplify the number of microtubules (Goshima, 2008).
Thus, Dgt proteins associate to form a stable complex. Based upon its role in increasing MT numbers in the spindle, it is proposed that this complex be called 'augmin' from the Latin verb augmentare, which means to increase. The role of augmin in increasing spindle MT density appears to be very important for building K-fibers and enabling chromatin-mediated MT nucleation to proceed toward the formation of a bipolar-shaped spindle, particularly when centrosome function is attenuated (Goshima, 2008).
The results suggest a model for how centrosome-, chromosome-, and spindle-based MT nucleation processes may cooperate to build mitotic spindles. After NEBD in somatic cells, the most obvious generation of new MTs occurs at centrosomes, producing astral MT arrays. MTs are also nucleated in the vicinity of chromosomes immediately after NEBD. These processes are critical for generating the first set of mitotic MTs, enabling the process of spindle assembly to begin. However, after the first sets of MTs are formed, it is proposed that augmin–γ-TuRC nucleates MT growth from existing MTs, thus providing a powerful mechanism for rapidly amplifying the number of MTs within the spindle to facilitate chromosome capture and K-fiber formation (Goshima, 2008).
It is postulated that kinetochore MTs, initially generated via the centrosome or chromatin pathway, are used as templates for binding augmin, which in turn recruits and activates γ-TuRC for MT nucleation. The outer γ-TuRC subunits appear to be critical for this specific spindle function of γ-tubulin because knockdown of these subunits produces a very similar phenotype to augmin knockdown, although evidence has not yet been obtained for a direct interaction between augmin and γ-TuRC. An intriguing possibility is that augmin may dock onto an MT in a manner that positions γ-TuRC to preferentially nucleate new MT growth with the same polarity as the parent MT (Goshima, 2008).
As an alternative to this model, augmin might increase the number of spindle MTs by activating an MT-severing enzyme, although this is unlikely because RNAi of katanin and other AAA ATPases (e.g., spastin) does not give rise to the same mitotic phenotype as Dgt or γ-TuRC RNAi. Another class of models is that augmin increases spindle MT density by either stabilizing, elongating, or transporting MTs that are nucleated at the kinetochore/chromatin and that augmin–γ-tubulin are not involved in MT nucleation within the spindle. Although this possibility cannot be ruled out, FRAP results showing the concomitant recovery of fluorescence at the center of the spindle and the spindle poles suggest that MTs are being formed throughout the spindle and not just near the chromatin. The notion of an MT amplification process is also supported by in vitro studies of the assembly of MT asters in X. laevis extract and accompanying computational simulations). The exact mechanism by which augmin increases MT density awaits future work, most decisively by in vitro reconstitution of the process with purified proteins (Goshima, 2008).
In addition to the mitotic spindle, there are other cellular MT networks in animals that are unlikely to be exclusively built by centrosome-based nucleation and assembly processes, such as axons or the meiotic spindle in oocytes. An MT-templated MT nucleation reaction could constitute an excellent means for generating new MTs while preserving the polarity of an existing MT array. Augmin is a candidate to play a role in this and numerous other cases where polarized noncentrosomal MTs are generated (Goshima, 2008).
gammaTubulin is a component of a previously isolated complex of
Drosophila proteins that contains at least two centrosomal microtubule-associated proteins (CP190
and CP60). Like CP190 and CP60, the gammaTubulin in extracts of early Drosophila embryos
binds to microtubules, although this binding may be indirect. Unlike CP190 and CP60, however, only
10-50% of the gamma-tubulin in the extract is able to bind to microtubules. gamma-tubulin
binds to a microtubule column as part of a complex, and a substantial fraction of this gamma-tubulin is tightly
associated with CP60. As neither alpha- nor beta-tubulin bind to microtubule columns, the
gammaTubulin cannot be binding to such columns in the form of an alpha:gamma or beta:gamma
heterodimer. These observations suggest that gammaRubulin, CP60, and CP190 are components of a
centrosomal complex that can interact with microtubules (Raff, 1993).
In Drosophila the male gamete is presumed to provide the active microtubule organizing center (MTOC). Zygotic centrosome assembly in fertilized Drosophila eggs was analyzed with the aid of an antiserum Rb188, previously shown to be specific for CP190, a 190 kDa centrosome-associated protein. The CP190 protein is detected in two discrete spots, associated with the anterior and posterior ends of the elongating nucleus of Drosophila spermatids. As the spermatids mature this labelling gradually disappears and is no longer visible in sperm dissected from spermathecae and ventral receptacles. gamma-Tubulin is also found in association with the posterior
end of the sperm nucleus during spermatogenesis, but is not detected in mature sperm. This suggests that CP190 and gamma-tubulin are not present in detectable quantities in fertilizing sperm. CP190 is not detected in association with the sperm nucleus of newly fertilized eggs removed from the uterus, whereas many CP190-positive particles are associated with microtubules of the sperm aster from anaphase I to anaphase II. It is thought that this CP190 is of maternal origin. These particles disappear during early telophase II; only one pair of CP190-positive spots remain visible at the microtubule focus of the sperm aster. These spots are associated with one aster through telophase, and then move away to form two smaller asters from which the first mitotic spindle is organized. Colchicine treatment suggests that at least some CP190 protein is an integral part of the centrosome rather than merely being transported along microtubules. Centrosomal localization of the CP190 antigen is prevented by incubation of the permeabilized zygote in 20 mM EDTA. At the end of mitosis the female MTOC is lost. The difference in behavior between the male and female MTOCs is probably due to the lack of important constituents in the female spindle that are necessary for the assembly of a centrosome which can reproduce and nucleate the first mitotic spindle. The most obvious difference between the male and female MTOCs is the presence of the paternal centrole at the center of the sperm aster; this centriole (and any associated pericentriolar material) could be the target for the assembly of the maternal components necessary for centrosome reproduction and nucleation of the mitotic spindle (Riparbelli, 1997).
CP190 is associated with the
centrosomes during mitosis, and relocates to chromatin during interphase. Staining of salivary gland chromosomes of third instar Drosophila larva, with antibodies specific to CP190,
indicates that the protein is associated with a large number of loci on these interphase polytene
chromosomes. The central region of the predicted amino acid sequence of the CP190 protein contains four
CysX2CysX12HisX4His zinc-finger motifs, similar to those described for several well characterised
DNA binding proteins. The data suggest that the function of CP190 involves cell cycle dependent
associations with both the centrosome, and with specific chromosomal loci (Whitfield, 1995).
The microtubule (MT) cytoskeleton is essential for
cell division and organization of the interphase cytoplasm. These functions are orchestrated by diverse and highly dynamic MT arrays generated by a variety
of mechanisms, including regulation of the polymerization dynamics of MTs, of proteins that interact with and organize MTs, and of MT nucleation. The latter mechanism is possible because the spontaneous nucleation of new tubulin polymers is kinetically
limiting, both in vitro when the polymerization of pure tubulin is initiated, and in vivo. Evidence for a kinetic barrier to MT nucleation in vivo
comes from analysis of repolymerization of MTs after cold
treatment or treatment with anti-MT agents. In many animal cells, regrowth initiates from the pericentriolar material (PCM) that surrounds the centrioles, demonstrating that the PCM promotes MT
nucleation. A major breakthrough in defining the molecular basis of the MT-nucleating activity of the PCM was the discovery of gamma-tubulin: gamma-tubulin is a member of the tubulin superfamily that localizes to
MT organizing centers and is found in all eukaryotes. Genetic
studies have demonstrated that gamma-tubulin is required for
normal cytoplasmic and spindle MT formation. In higher eukaryotes, soluble gamma-tubulin exists primarily
in a large complex (between 25 and 32 S). This complex was purified from Xenopus
egg extracts and shown to nucleate MTs in vitro. This complex, called the gammaTuRC (gamma-tubulin
ring complex), consists of about eight proteins in addition
to gamma-tubulin and has the appearance of an open ring with approximately the same diameter as a MT (Zheng, 1995). Rings of this diameter have also been observed in
the PCM of centrosomes isolated from Drosophila embryos (Moritz, 1995a). In Drosophila, immunoelectron microscopy has confirmed the presence of clusters of gamma-tubulin in
the ring structures and at the base of MTs nucleated by the PCM (Moritz, 1995b). Cumulatively, these results
suggest that the gammaTuRC is a highly conserved structure responsible for the MT-nucleating activity of the PCM (Oegema, 1999 and references).
The gamma-tubulin in S. cerevisiae is the most divergent of all
gamma-tubulins. It is only ~35-40% identical to the other
known gamma-tubulins, all of which are at least 65% identical to
one another. In S. cerevisiae, the
only known soluble gamma-tubulin-containing complex is ~6 S
and contains three proteins: gamma-tubulin, and two related
proteins, Spc97p and Spc98p (Geissler, 1996 and Knop, 1997b). Immunoprecipitation experiments with tagged proteins suggest that the S. cerevisiae complex contains one molecule of Spc97p, one
molecule of Spc98p, and two or more molecules of gamma-tubulin (Knop, 1997a and Knop, 1997b). The yeast
gamma-tubulin 6 S complex is thought to be anchored to the cytoplasmic side of the spindle pole body through the interaction of Spc97p and Spc98p with Spc72p (Knop, 1998), and to the nuclear side of the spindle
pole body through interaction with the NH2 terminus of
Spc110p (Knop, 1997b). To date, in vitro MT-nucleating activity for the yeast complex has not been
demonstrated. Therefore, it remains unclear whether the yeast gamma-tubulin complex nucleates MTs directly, or whether
it assembles into a larger, perhaps gammaTuRC-like structure
at the spindle pole body. Interestingly, homologs of
Spc97p and Spc98p in humans (hGCP2 and hGCP3/
HsSpc98; Murphy, 1998, Tassin, 1998) and in
Xenopus (Xgrip109; Martin, 1998) colocalize with
gamma-tubulin at the centrosome and cosediment with gamma-tubulin on sucrose gradients, indicating that they are components
of the large gamma-tubulin-containing complexes present in these organisms (Oegema, 1999 and references).
Understanding the role of gamma-tubulin in MT nucleation is
a challenging endeavor. Low cellular concentrations make
purification from native sources difficult, and the complexity of the protein complexes that contain gamma-tubulin limits
expression-based studies. Analysis of MT nucleation is
further complicated by the following: the complex structure of the MT lattice; the
large number of tubulin molecules potentially involved in the formation of a nucleus, and the potential role of beta-tubulin
GTP hydrolysis in suppressing nucleation. This difficulty is reflected by the fact that the mechanism of spontaneous nucleation of purified tubulin remains poorly understood (Oegema, 1999 and references).
Central to an understanding of the mechanism of MT nucleation by gamma-tubulin-containing complexes will be an understanding of the relationship between gamma-tubulin and other members of the tubulin superfamily. One important aspect of
this relationship is the nature of the contacts gamma-tubulin
makes with itself and with alpha- or beta-tubulin. A second important aspect is how gamma-tubulin compares to other members of the tubulin family in its ability to bind and hydrolyze GTP. If gamma-tubulin binds a guanine nucleotide, it will
be important to determine whether nucleotide exchange
and hydrolysis contribute to its ability to assemble, disassemble, nucleate, or release MTs, or whether the bound
nucleotide has a structural role, as is the case for alpha-tubulin. The functional organization of the gammaTuRC in Drosophila has been addressed by purifying and analyzing gamma-tubulin-containing complexes from Drosophila embryo extracts.
In Drosophila, there are two related gamma-tubulin-containing
complexes. These have been named gamma-tubulin small complex (gammaTuSC;
~280,000 D) and Drosophila gammaTuRC (~2,200,000 D). In addition to gamma-tubulin, the
gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family.
The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six
additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts, the Drosophila gammaTuRC can
nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron
microscopy reveals a modular structure with ~13 radially arranged structural repeats. The
gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting
that assembly into a larger complex enhances nucleating activity.
The larger complex can be collapsed into the
smaller complex by treatment with high salt. This condensation suggests that the small complex is a structural subunit of the large complex (Moritz, 1998). Both complexes have now been purified and it has been shown that the large Drosophila complex nucleates MTs much more potently than the small
complex. Analysis of the nucleotide content of
the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering
gamma-tubulin an unusual member of the tubulin superfamily (Oegema, 1999).
To determine the protein compositions of the gammaTuSC and
gammaTuRC, complexes were fractionated on
a 5% to 40% sucrose gradient. The protein
profile of the Drosophila gammaTuRC is reminiscent of the Xenopus gammaTuRC. Therefore, by analogy to the
Xgrips (Martin, 1998), Drosophila gammaTuRC
proteins have been named Dgrips and have been designated by their apparent molecular weights. Like the Xenopus gammaTuRC, the Drosophila
gammaTuRC is composed of two high molecular mass proteins
(Dgrip163 and Dgrip128), two prominent proteins near
100 kD (Dgrip91 and Dgrip84), and a group of three or
four proteins with molecular masses near 75 kD (Dgrip75s). It is not clear
whether actin is a specific component of gammaTuRC, or if it
fortuitously copurifies. Depending on the purification protocol, varying amounts of alpha- and beta-tubulin copurify with
Xenopus gammaTuRC (Zheng, 1995). In contrast, no
alpha- or beta-tubulin copurifying with Drosophila gammaTuRC could be detected.
Consistent with the idea that gammaTuSC is a structural subunit
of gammaTuRC, gammaTuSC is composed of the three most prominent proteins in gammaTuRC: gamma-tubulin, Dgrip84, and Dgrip91 (Oegema, 1999).
To characterize the molecular nature of gammaTuSC, its non-gamma-tubulin components, Dgrip84
and Dgrip91 were cloned and sequenced. Dgrip84 and Dgrip91 are homologous to
each other and to the Spc97/98p family of proteins identified in S. cerevisiae. This family also includes two proteins
identified in humans, hGCP2 and hGCP3 (Murphy, 1998). The homology between the Drosophila proteins
and the other members of this family extends over the entire length of the proteins. In comparisons of Dgrip84 and Dgrip91 with the corresponding
human proteins, a one-to-one correspondence emerges.
Dgrip84 is 32% identical (46% similar) to hGCP2 and
only 21% identical (32% similar) to hGCP3; in contrast,
Dgrip91 is 31% identical (45% similar) to hGCP3 and
only 24% identical (37% similar) to hGCP2. These results suggest that Dgrip84 and hGCP2, and Dgrip91 and
hGCP3 may be functionally homologous pairs. Since an estimate of the molecular mass of purified
gammaTuSC from sucrose gradient sedimentation and gel filtration is 280,000 D, it is suspected that gammaTuSC contains 1 molecule of Dgrip91, 1 molecule of Dgrip84, and 2 molecules of gamma-tubulin. Interestingly, this corresponds to
estimates of the stoichiometry of proteins in the S. cerevisiae 6 S gamma-tubulin complex (Knop, 1997a and b). If it is assumed that gammaTuRC contains only
one molecule of each non-gammaTuSC component, then gammaTuRC would contain approximately six gammaTuSCs (Oegema, 1999).
If gamma-tubulin in Drosophila embryos primarily exists associated with Dgrip84 and Dgrip91 in either gammaTuSC or
gammaTuRC, these three proteins would be expected to cofractionate on sucrose gradients of embryo extract and to
colocalize in embryos. To test this hypothesis, rabbit polyclonal antibodies were produced that recognize Dgrip84 and Dgrip91. As expected, both
Dgrip84 and Dgrip91 comigrate with gamma-tubulin in gammaTuSC
and gammaTuRC when embryo extract is fractionated on sucrose gradients. In addition, the localizations of Dgrip84 and Dgrip91 in Drosophila embryos are
indistinguishable from those of gamma-tubulin. Each antibody
recognizes the centrosome throughout the cell cycle and
shows some spindle staining during mitosis with enrichment at the spindle poles, regardless of its cognate antigen. It is proposed that Drosophila gamma-tubulin is stably associated with Dgrip91/84. Interestingly, no evidence is found for a non-gamma-tubulin associated pool of either
Dgrip84 or Dgrip91 (Oegema, 1999).
The homology between gamma-, alpha-, and beta-tubulins extends into
domains that are involved in GTP binding by alpha- and
beta-tubulin. Thus, it is tempting to speculate
that gamma-tubulin can bind, and possibly hydrolyze, GTP. To
determine if gamma-tubulin binds guanine nucleotide, gamma-tubulin-containing complexes were immunoisolated in the absence
of GTP. The isolated complexes, either before or after sucrose gradient sedimentation, were incubated with
[alpha-32P]GTP and UV cross-linked. In the peptide-eluted
complexes, gamma-tubulin is the only protein that cross-links
to GTP. Furthermore, gamma-tubulin in both the
gammaTuRC and gammaTuSC cross-links to GTP. Competition experiments show that the cross-link can be competed by addition of excess cold GTP, GDP, and GTPgammaS
but not GMP-PNP, ATP, or CTP (Oegema, 1999).
To characterize the nucleotide binding properties of
gamma-tubulin, the nucleotide content of gamma-tubulin in gammaTuSC was compared to that of similarly treated alphabeta-tubulin dimer. When gammaTuSC is analyzed, the nucleotide recovered from gammaTuSC incubated in GDP buffers is exclusively GDP. Approximately 0.7 mol GDP is
recovered per mole of gamma-tubulin.
The exclusive presence of GDP could be explained at least
three ways: (1) the guanine nucleotide binding site on
gamma-tubulin subunits of gammaTuSC is freely exchangeable; (2) GDP is locked nonexchangeably into gamma-tubulin subunits of
gammaTuSC, analogous to GTP bound at the N-site in alpha-tubulin; or (3) GDP is locked nonexchangeably into gammaTuSC as
the product of earlier GTP hydrolysis, much like beta-tubulin
bound GDP within the body of a polymerizing MT (Oegema, 1999).
To distinguish between these possibilities,
gammaTuSC was isolated from GTP-containing buffer. Surprisingly, a greatly reduced amount of nucleotide was recovered.
Only 0.2 mol guanine nucleotide was recovered per mole
of gamma-tubulin, indicating that ~80% of the gamma-tubulin was
empty at its nucleotide binding site. The low recovery of
guanine nucleotide bound to gammaTuSC isolated from GTP
buffer indicates that GDP is bound exchangeably to
gamma-tubulin in gammaTuSC. This result also argues against the
theory that the GDP bound to gamma-tubulin in gammaTuSC, isolated from GDP buffer, is being generated by earlier GTP
hydrolysis. The recovery of nearly 1 mol GDP per mole
of gamma-tubulin from GDP buffer and the nearly equivalent
amounts of GTP and GDP in the 0.2 mol nucleotide recovered per mole of gamma-tubulin from GTP buffer, despite a
GTP/GDP ratio of greater than 30 before desalting, strongly suggest
that gamma-tubulin in gammaTuSC has an exchangeable guanine nucleotide binding site that has a much higher affinity for
GDP than GTP (Oegema, 1999).
An important issue with respect to the in vivo roles of
gammaTuSC and gammaTuRC is their relative MT nucleating activity.
The fact that S. cerevisiae does not appear to contain a
gammaTuRC-like complex raises the question of whether Sc
gammaTuSC has nucleating activity or whether it must assemble
into a larger structure at the spindle pole body to become
active. Conversely, in metazoa it is possible that gammaTuRC is
a storage form for gamma-tubulin and it could be gammaTuSC that
nucleates MTs at centrosomes (Knop, 1997b).
For the Drosophila complexes, per mole of gamma-tubulin, gammaTuRC is ~25 times
more active than gammaTuSC in promoting nucleation. Combining these data with stoichiometry measurements, it is estimated that per mole of complex gammaTuRC is ~150
times more active than gammaTuSC, suggesting that organization of gammaTuSC into gammaTuRC facilitates MT nucleation activity (Oegema, 1999).
In agreement with others working on Drosophila
centrosomal proteins, the names for DMAP190 and DMAP60 have been changed to CP190 and CP60,
respectively, to give these proteins a consistent nomenclature. In Drosophila, CP190 is a microtubule-associated protein that is localized to the centrosome. Affinity chromatography was used to identify proteins that interact with CP190, allowing the identification of CP60. Like CP190, CP60
interacts with microtubules and is localized to the centrosome. The two proteins associate as part of a
multiprotein complex. The
amino acid sequence of CP60 is not homologous to any protein in the database, although it contains six
consensus sites for phosphorylation by cyclin-dependent kinases. As judged by in situ hybridization, the
gene for CP60 maps to chromosomal region 46A. Antibodies that recognize CP60 reveal that it
is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is
maximal during anaphase and telophase, and then drops dramatically sometime between late telophase and early
interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains
a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of
mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase.
CP60 isolated from Drosophila embryos is highly phosphorylated, and dephosphorylated CP60 is a good
substrate for cyclin B/p34cdc2 kinase complexes. A second kinase activity capable of phosphorylating CP60
is present in the CP60/CP190 multiprotein complex. CP60 binds to purified
microtubules, and this binding is blocked by CP60 phosphorylation (Kellogg, 1995).
CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner, moving
between the nucleus (during interphase), and the centrosome (during mitosis). A single bipartite 19-amino acid nuclear localization signal was detected
that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124
amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show
weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization
sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion
proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell
cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the
nucleus during interphase. The domain responsible for microtubule binding overlaps the
domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also
localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Both CP190 and CP60
are able to attain and maintain their centrosomal localization in the absence of microtubules (Oegema, 1995).
Both the nucleus and the centrosome are complex, dynamic structures whose
architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two
Drosophila proteins of unknown function that shuttle between centrosomes and nuclei
in a cell cycle-dependent manner. These two proteins are associated in vitro, and
localize to centrosomes in a microtubule independent manner. Fluorescently labeled, bacterially expressed CP190 and CP60 were injected into living Drosophila
embryos and their behavior followed during the rapid syncytial blastoderm divisions
(nuclear cycles 10-13). CP190 and CP60 cycle between nuclei and centrosomes
asynchronously with the accumulation of CP190 leading that of CP60 both at
centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately
following nuclear envelope breakdown, CP190 localizes to centrosomes where it
remains until telophase, thereafter accumulating in reforming nuclei. Unlike CP190,
CP60 accumulates at centrosomes primarily during anaphase, where it remains into
early interphase. During nuclear cycles 10 and 11, CP60 accumulates in nuclei
simultaneous with nuclear envelope breakdown, suggesting that CP60 binds to an
unknown nuclear structure, which persists into mitosis. During nuclear cycles 12 and 13,
CP60 accumulates gradually in nuclei during interphase, reaching peak levels just
before nuclear envelope breakdown. Once in the nucleus, both CP190 and CP60
appear to form fibrous intranuclear networks that remain coherent even after nuclear
envelope breakdown. The CP190 and CP60 networks do not co-localize extensively
with one another or with DNA. This work provides direct evidence, in living cells, of a
coherent protein network that may represent a nuclear skeleton (Oegema, 1997).
The gamma-tubulin ring complex (gammaTuRC) is a protein complex of relative
molecular mass ~2.2 x 106 that nucleates microtubules
at the centrosome. Electron-microscopic tomography and metal shadowing has been used
to examine the structure of isolated Drosophila gammaTuRCs and the
ends of microtubules nucleated by gammaTuRCs and by centrosomes. The gammaTuRC is a lockwasher-like structure made up of repeating subunits,
topped asymmetrically with a cap. A similar capped ring is also visible at
one end of microtubules grown from isolated gammaTuRCs and from centrosomes.
Antibodies against gamma-tubulin label microtubule ends, but not walls, in
centrosomes. These data are consistent with a template-mediated mechanism
for microtubule nucleation by the gammaTuRC (Moritz, 2000).
The reconstructions that were obtained lead to the proposal of models for the
arrangement of gammaTuRC proteins within the complex as well as for points
of contact between the gammaTuRC and the microtubule. The repeating subunits in the ring wall are
most probably made up of gamma-tubulin, Dgrip84 and Dgrip91. The latter two
proteins (or their orthologs yeast Spc97 and Spc98, and human GCP2 and GCP3/HsSpc98p) are known to interact with each other and with gamma-tubulin. In Drosophila and S. cerevisiae, these
proteins have been found to form a small complex that contains two gamma-tubulins
and one molecule each of Dgrip84 (or Spc97) and Dgrip91 (or Spc98). In addition, stoichiometric studies of the Drosophila
proteins (and some of their Xenopus counterparts)
in the gammaTuRC show that these three proteins are the most abundant in the
complex, indicating that they may comprise the ring wall,
where many repeating subunits are visible. Moreover, it is likely that one
of the paired, V-shaped subunits corresponds to one gamma-tubulin small complex
(gammaTuSC in Drosophila). Since the height of each column in the subunit (~10 nm) is too great
for it to be made up of a single molecule of one of these proteins, it is proposed
that a tandem arrangement of Dgrip91 or Dgrip84 with gamma-tubulin exists. In this model, the gamma-tubulins
are located most proximally to the face of the ring that does not contain
the cap, and are therefore available to bind to alpha/beta-tubulin, thereby
promoting microtubule growth (Moritz, 2000).
The gamma-tubulin ring complex (gammaTuRC) is important for microtubule nucleation from the centrosome. In addition to gamma-tubulin, the Drosophila gammaTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips)
75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small gamma-tubulin complex gammaTuSC) and the gammaTuRC, while the remaining subunits are found only in the gammaTuRC. To study gammaTuRC assembly
and function, gammaTuSC was reconstituted using the baculovirus expression system. Using the reconstituted gammaTuSC, it has been shown that this subcomplex of the gammaTuRC has microtubule binding and capping activities. Two new gammaTuRC subunits, Dgrips128 and 163, have been characterized and they are shown to be centrosomal proteins. Sequence comparisons among all known gammaTuRC subunits reveal two novel sequence motifs, which have been named grip motifs 1 and 2. Dgrips128 and 163 can each interact with gammaTuSC. However, this interaction is insufficient for gammaTuRC assembly (Gunawardane, 2000).
With the reconstitution of the gammaTuSC, an important step has been made toward understanding the assembly and function of the gammaTuRC. By both functional and biochemical criteria, the reconstituted gammaTuSC is identical to the endogenous gammaTuSC. In addition to its nucleating activity, the gammaTuSC can also bind and cap the minus ends of MTs. This observation is consistent with the finding that the monomeric gamma-tubulin and gammaTuRC can also bind and cap the minus ends of MTs. Compared with gammaTuRC, gammaTuSC is a weak MT nucleator. Consistent with its weak nucleating activity, the capping activity of gammaTuSC is significantly less than that of the gammaTuRC. For example, ~50% of MTs nucleated in the presence of gammaTuRC were capped, while under the same assay conditions, gammaTuSC could only cap up to 20% of MTs. One explanation for this weak nucleating and capping activity of gammaTuSC is that gammaTuSC contains only two gamma-tubulin molecules, while the intact gammaTuRC contains ~12 gamma-tubulin molecules (Gunawardane, 2000).
The existence of the conserved sequence motifs in all five of the grips suggests that gammaTuRC assembly may be mediated by conserved structural surfaces defined by these motifs. A provocative idea is that grip motif 2, which is present in all five grips, is involved in interacting with a common protein in the gammaTuRC (e.g., gamma-tubulin). Consistent with these ideas, gamma-tubulin coimmunoprecipitates with each of the five Dgrips when coexpressed in pairs. Furthermore, using similar methods, it has been found that the Dgrips also interact with each other. It will be important to study the nature of these interactions and test whether they are mediated by the grip motifs (Gunawardane, 2000).
Alternatively, the grip motifs could be involved in binding the gammaTuRC to its centrosomal docking site. Using in vitro centrosome assembly assays in Xenopus egg extracts, it has been shown that the removal of Xgrip210 (Dgrip163 homolog) blocks the localization of Xgrip109 (Dgrip91 homolog) to the centrosome and vice versa. This suggests that the grip motifs present in Xgrips109 and 210 are not sufficient for the binding of individual grips to the centrosome. Therefore, it is unlikely that the individual grip motifs per se are sufficient to mediate the recruitment and binding to the centrosomes. Instead, a certain combination of the grip motifs may be required for centrosomal docking to take place, and such a structural surface may only occur in the intact gammaTuRC (Gunawardane, 2000).
Based on the structural features of the gammaTuRC, four possible models are proposed for gammaTuRC assembly. In the first model, the cap structure of the gammaTuRC acts as a scaffold onto which multiple gammaTuSCs assemble to form a ring. In this assembly pathway, the formation of the ring requires preassembly of the cap structure. In the second model, multiple gammaTuSCs oligomerize and individual cap subunits add onto this oligomer to form the gammaTuRC. In this model, prior assembly of a cap structure is not required and gammaTuSC polymerization drives the assembly process. The third model features the preassembly of both a cap structure and gammaTuSC oligomers. In this model, the gammaTuSC oligomers are stabilized by the preformed cap structure to form a gammaTuRC. Finally, the fourth model predicts that the gammaTuRC is assembled sequentially from several distinct intermediates (Gunawardane, 2000).
The majority of the reconstituted and purified gammaTuSC migrates as a 10 S complex on sucrose gradients. However, a small fraction of the gammaTuSC appears to oligomerize and migrate faster than the 10 S complex. This observation suggests that oligomerization of gammaTuSC could contribute toward gammaTuRC assembly. This success in gammaTuSC reconstitution should allow the further testing of conditions that promote gammaTuSC oligomerization and aid the study of gammaTuRC assembly (Gunawardane, 2000).
Although coexpressing Dgrips128 and 163 with gammaTuSC does not promote gammaTuSC oligomerization or gammaTuRC assembly, both these proteins can interact with gammaTuSC independent of each other. These interactions may give rise to the assembly intermediates, as suggested by the sequential pathway of gammaTuRC assembly. In addition, in vitro assays using Xenopus egg extract showed that the Dgrip163 homolog Xgrip210 is essential for gammaTuRC assembly. Based on these observations, it is suggested that Dgrips128 and 163 are essential but not sufficient for gammaTuRC formation. If all gammaTuRC subunits are needed for its assembly, the identification and expression of Dgrip75s (the remaining subunits of gammaTuRC) should permit the reconstitution of the gammaTuRC and the testing of the various models for gammaTuRC assembly (Gunawardane, 2000).
The gamma-tubulin ring complex (gammaTuRC), purified from the cytoplasm of vertebrate and invertebrate cells, is a microtubule nucleator in vitro. Structural studies have shown that gammaTuRC is a structure shaped like a lock-washer and topped with a cap.
Microtubules are thought to nucleate from the uncapped side of the gammaTuRC. Consequently, the cap structure of the gammaTuRC is distal to the base of the microtubules, giving the end of the microtubule the shape of a pointed cap. The cloning and characterization of a new subunit of Xenopus gammaTuRC, Xgrip210, is reported. Xgrip210 is a conserved centrosomal protein that is essential for the formation of gammaTuRC. Using immunogold labeling, it has been found that Xgrip210 is localized to the ends of microtubules nucleated by the gammaTuRC and that its localization is more distal, toward the tip of the gammaTuRC-cap structure,
than that of gamma-tubulin. Immunodepletion of Xgrip210 blocks not only the assembly of the gammaTuRC, but also the recruitment of gamma-tubulin and its interacting protein, Xgrip109, to the centrosome. These results suggest that Xgrip210 is a component of the gammaTuRC cap structure that is required for the assembly of the gammaTuRC (Zhang, 2000).
The spindle pole localization of gamma-tubulin was compared in wild type and acentriolar cultured Drosophila cells using polyclonal
antibodies specifically raised against the carboxy terminal amino acid sequence of Drosophila gamma-tubulin-1.
During interphase, gamma-tubulin is present in the centrosome of wild type cells and accumulates around this organelle in a cell
cycle dependent manner. In contrast, no such structure is observed in acentriolar cells. Wild type mitoses are homogeneously
composed of biconical spindles, with two centrosome-associated gamma-tubulin spots at the poles. The mitotic apparatuses observed
in the acentriolar cells are heterogeneous; multipolar mitoses, bipolar mitoses with a barrel-shaped spindle and bipolar mitoses with
biconical spindles were observed. In acentriolar cells, gamma-tubulin accumulation at mitotic poles is dependent on spindle
microtubule integrity. Most acentriolar spindles present a dispersed gamma-tubulin labeling at the poles. Only well polarized and
biconical acentriolar spindles show a strong gamma-tubulin polar spot. Finally, acentriolar mitotic poles are not organized around
true centrosomes. In contrast to wild type cells, in acentriolar cells the Bx63 centrosome-associated antigen is absent and the
gamma-tubulin containing material dispersed readily following microtubule disassembly. These observations confirm that
gamma-tubulin plays an essential role in the nucleation of microtubules even in the absence of mitotic polar organelles. In addition the
data suggest that the mechanisms involved in the bipolarization of wild type and acentriolar mitoses are different, and that centrioles
play a role in the spatial organization of the nucleating material containing gamma-tubulin (Debec, 1995).
The protein serine/threonine phosphatase 4 (PP4), which localises to centrosomes/spindle pole bodies in human cells, is shown to exhibit a similar localization in Drosophila cells and embryos and possess a highly conserved (from humans to invertebrates) amino acid sequence (91% identical). A homozygous Drosophila melanogaster strain mutant in the PP4 gene at 19C1-2 has been produced using P element mutagenesis. This strain, termed centrosomes minus microtubules (cmm), has reduced amounts of PP4 mRNA (~25% of normal PP4 protein in early embryos) and exhibits a semi-lethal phenotype with only 10% viability under certain conditions. Reversion mutagenesis shows that the phenotype is due to the presence of the P element in the PP4 mRNA. In early cmm embryos, nuclear divisions become asynchronous and large regions containing centrosomes with no well defined radiating microtubules are visible. In such areas, most nuclei arrest during mitosis with condensed DNA, and mitotic spindle microtubules are either absent, or aberrant and unconnected to the centrosome. A reduction in the staining of gamma-tubulin at centrosomes in cmm embryos suggests a conformational change or relocation of this protein, which is known to be essential for initiation of microtubule growth. The total level of gamma-Tubulin is identical in cmm and wild-type embryo extracts. Therefore it appears that there must be a relocation or conformational change of gamma-Tubulin in cmm embryos. To confirm that centrosomes are still present in severely abnormal embryos, the localization of CP190 was examined and found to be similar in the wild-type and cmm embryo, even when gamma-Tubulin staining was very weak in the latter. These findings indicate that PP4 is required for nucleation, growth and/or stabilization of microtubules at centrosomes/spindle pole bodies (Helps, 1998).
The Drosophila gene discs degenerate-4 (dd4) has been cloned and found to encode a component of the gamma-tubulin ring complex (gammaTuRC) homologous to Spc98 of budding yeast. This provides the first opportunity to study decreased function of a member of the gamma-tubulin ring complex, other than gamma-tubulin
itself, in a metazoan cell. gamma-tubulin is no longer found at the centrosomes
but is dispersed throughout dd4 cells and yet bipolar
metaphase spindles do form, although these have a dramatically
decreased density of microtubules. Centrosomin (CNN) remains in broad
discrete bodies but only at the focused poles of such spindles, whereas
Asp (abnormal spindle protein) is always present at the presumptive minus
ends of microtubules, whether or not they are focused. This is
consistent with the proposed role of Asp in coordinating the nucleation
of mitotic microtubule organizing centers. The centrosome associated
protein CP190 is partially lost from the spindle poles in dd4
cells supporting a weak interaction with gamma-tubulin, and the displaced
protein accumulates in the vicinity of chromosomes. Electron microscopy
indicates not only that the poles of dd4 cells have irregular
amounts of pericentriolar material, but also that they can have
abnormal centrioles. In six dd4 cells subjected to serial
sectioning, centrioles were missing from one of the two poles. This
suggests that in addition to its role in nucleating cytoplasmic and
spindle microtubules, the gammaTuRC is also essential to the structure of
centrioles and the separation of centrosomes (Barbosa, 2000).
The major microtubule organizing center (MTOC) in
animal cells is the centrosome, which nucleates the slowly growing
minus ends of microtubules allowing the plus ends to extend into the
cytoplasm. In most animal cells, centrosomes are essential for
definition of the interphase MT arrays, for determination of cell
polarity, and for the formation and function of the spindle in mitosis.
There are two main components of the centrosome: a pair of centrioles
comprising cylinders of nine triplet microtubules and the
pericentriolar material (PCM) that appears to provide nucleation
centers for cytoplasmic and spindle microtubules. Little is known about
the organization of the PCM, although both pericentrin and gamma-tubulin
have been described as forming a protein complex organized into a lattice
like structure. gamma-tubulin is a conserved member of the tubulin family found at MTOCs, including animal cell centrosomes, and the equivalent organelles of
yeasts, the spindle pole bodies (SPBs). The gamma-tubulin of
S. cerevisiae forms a 6S complex with Spc98p and Spc97p,
associated with both the inner and outer plaques of the SPB. Temperature sensitive mutants of its structural gene, tub4, show defects in microtubule nucleation
at the newly formed SPB as well as in the assembly of a mitotic spindle. The SPC98 gene was identified as a dosage-dependent suppressor of the
tub4-1(ts) allele. Its gene product
appears essential for mitotic spindle formation because cells harboring
the temperature sensitive allele spc98-1 or over expressing
wild-type protein, duplicate and separate their SPBs but form a
defective mitotic spindle. The gene encoding the
other main component of this complex, SPC97, was isolated as a
suppressor of the spc9-2(ts) mutant. Its
temperature sensitive alleles show phenotypes similar to tub4
and spc98 mutants as well as defects in SPB duplication. Spc98p docks the Tub4p complex to the inner plaque of the
SPB through the N terminus of Spc110p, a protein that forms a bridge between the inner and central plaques. The Tub4 complex is formed in the cytoplasm. It is transported to the nucleus via the nuclear localization signal (NLS) present in Spc98p. Spc98p at the inner plaque of the SPB is phosphorylated
in a cell cycle-dependent manner, whereas Spc98p in the outer plaque
does not appear to undergo such modification (Barbosa, 2000).
In higher eukaryotes gamma-tubulin occurs in a 25S-32S complex that has
been shown by electron microscopy to have a ring shape leading to the
name gamma-tubulin ring complex (gammaTuRC). The Xenopus complex comprises seven proteins: alpha-, beta-, and gamma-tubulin, and additional proteins of 75, 109, 133, and
195 kD. The 109 kD Xenopus protein (Xgrip109) is a homolog of yeast Spc98p. It interacts directly with gamma-tubulin and is essential for microtubule nucleation. The major human and Drosophila gammaTuRCs have very similar protein profiles. The 100 and 101 kD human proteins hGCP2 and hGCP3 correspond to Spc97p and Spc98p, respectively. In Drosophila, a second smaller 240 kD gamma-tubulin
complex has been described comprising only
gamma-tubulin and the Spc97/98 homolog Dgrip84 and
Dgrip91. It is proposed that this is assembled into the
complete 3 MDa gamma-TuRC, which contains multiple copies of the
heterotrimer plus ancillary proteins (Barbosa, 2000 and references therein).
A model of the gammaTuRC in Drosophila suggests that
the complex is assembled in the cytoplasm from the heterotrimer subunits and recruited onto the centrosome, where it nucleates microtubules. In mitosis it
functions in concert with Asp, or a protein with equivalent function in
other organisms, to organize the spindle microtubules (Avides, 1999). This implies that the centrosome might be essential for
MT nucleation and therefore for the formation of spindles. However, in
the female meiotic divisions of Drosophila the spindles form
in the absence of centrioles and without
detectable concentration of gamma-tubulin at the poles. Moreover, loss of centrosomes has been
observed from the spindle poles in the syncytial embryos of several
mutants, and although these generally lead to the accumulation of
mitotic defects, several rounds of mitosis can take place on such
spindles. These observations, together with
the ability to build spindles without centrosomes in vitro that are
able to undertake metaphase and anaphase, has been taken to mean that
centrosomes might be dispensable in the formation of a functional
spindle in some systems. Recent observations that a functional spindle can still form in mammalian cells after laser ablation of the centrosomes now reinforce this idea (Barbosa, 2000 and references therein).
Because dd4 encodes a component of the gamma-tubulin ring
complex, the organization of the mitotic spindle and its poles were examined in cells from the central nervous system of dd4 mutants. The localization of gamma-tubulin was examined in relation to centrosomin (CNN), another component of the Drosophila centrosome. In mitotic cells
from wild-type brains, these two proteins colocalize to the two
centrosomes. In cells of all three dd4 mutant alleles, gamma-tubulin staining can still be detected; however, it is no longer found in a well-defined body but
rather is distributed throughout the cell. In contrast,
distinct CNN-containing bodies can be seen in every mitotic cell.
However, whereas wild-type cells invariably contained two such bodies (the functional centrosomes), some mutant cells contained only one body
stained with CNN, while others contained two (Barbosa, 2000).
In cells stained to reveal the spindle microtubules, the
CNN-containing bodies always appear to be associated with a
microtubule organizing center. In wild-type cells, these are spherical
centrosomes at the spindle poles and astral microtubules. In the dd4 mutant cells, the
CNN staining bodies are often less tightly defined structures and astral
microtubules are not seen. In some cells the CNN containing body
appears to have fragmented, and a satellite body can be seen near
the main pole. In those cells having only one
CNN-staining body, prominent arrays of microtubules extend between
this pole and the chromosomes. Conversely, microtubules
extending from the chromosomes to the pole lacking the CNN body
exhibit reduced staining, and in some cells this pole
appears not to have organized microtubules (Barbosa, 2000).
In addition to the gamma-tubulin ring complex, the Asp protein is also
known to be required to nucleate asters of microtubules (Avides, 1999). In wild-type mitotic cells, the Asp protein is found on the
face of the centrosome that makes contact with spindle microtubules. This
close juxtaposition of gamma-tubulin and Asp is no longer seen in
dd4cells in which the gamma-tubulin is dispersed, but Asp
maintains a punctate distribution. This punctate staining can be
clustered around the spindle poles or clustered in one area and
scattered throughout the remaining part of the cell,
but is never diffuse as is gamma-tubulin. Immunostaining to reveal
microtubules shows that Asp protein is always found at the poles of
bipolar spindles, either as a well organized body, but more usually in clustered aggregates. In spindles that had only one focused
pole, individual bundles of the microtubules could be seen to extend
both from this focus and from small punctate caps of Asp at the
unfocused pole towards the chromosomes (Barbosa, 2000).
CP190 is an abundant protein which, together with its partner CP60,
associates with mitotic centrosomes in Drosophila cells. Although frequently used as a marker to follow centrosome behavior, its function remains unknown. In wild-type cells, CP190 is found associated with the centrosomes at the spindle pole.
In the weakest allele, dd4S, some of the antigen is
lost from the poles and appears in the central part of the spindle in
the region occupied by the mitotic chromosomes. In the
stronger alleles, only a small proportion of CP190 remains at the
poles; the remainder is clustered around the chromosomes. Thus, intact gamma-tubulin ring complex appears to be required at the
poles in order to maintain the polar association of the CP190 protein (Barbosa, 2000).
In addition to being present in the PCM, gamma-tubulin is also found
within the centrioles themselves and appears to be required for
centriolar function. To
determine whether dd4 mutants showed any irregularity of
centriolar structure, the ultrastructure of centrosomes was examined by
electron microscopy of serial sections of cells from the larval central
nervous system. These data consist of complete sets of
serial sections through some four wild-type prometaphase cells with
chromosomes undergoing congression, one wild-type cell at metaphase,
and six dd41mutant cells that are in a
metaphase-like state. The centrosomes have
well defined centrioles showing typical arrays of triplet microtubules surrounded by the electron-dense PCM. Arrays of spindle microtubules extend from
these centrosomes towards condensed chromosomes.
These microtubules occur in bundles that make contact with well defined
kinetochore plates on the chromosomes. All of the dd4 cells examined had two opposing poles, generally broad, but
secondary poles were often present. Centrioles were present only at one
of the two main poles in all six of the cells, but there were up to
four of them; they varied in size and were not consistently arranged as
perpendicular pairs. The amount of PCM varied from one pole to another
showing no correlation with the pattern of centrioles. The density of spindle microtubules in dd4 cells is strikingly reduced compared to
the wild-type spindle, consistent with the
impression gained from immunostaining. The spindle
microtubules make poor contact with the mitotic chromosomes and, in
contrast to wild-type cells, it was difficult to see organized plate-like kinetochores on dd4 chromosomes (Barbosa, 2000).
It is of interest to compare phenotypes of mutations in dd4 with mutations in the gamma-tubulin genes. Drosophila has
two genes for gamma-tubulin; the one at 23C is expressed in a variety of
tissues including brains, imaginal discs and testes, whereas expression
of the second at 37C is restricted to ovaries and embryos. Like the dd4 mutants, cells from gamma-tub23C brains display abnormally high levels of
chromosome condensation, spindles with defective or absent poles, and
polyploidy. However, whereas the mitotic index of dd4 cells is
dramatically elevated, the mitotic index of gamma-tub23C cells
is reduced relative to wild-type and anaphase figures are very rare. The reasons for these differences are not clear.
It is as though reduction in levels of the 23C gamma-tubulin lead to a
limited numbers of chromosome duplication cycles in the absence of
mitosis, but this is followed by interphase cell cycle arrest. In
contrast, the dramatic increase in metaphase figures in dd4 mutants resembles a more typical spindle checkpoint arrest. The
frequency of anaphases, not dissimilar in total number to wild type,
suggests that cells can evade this checkpoint at some frequency, as has
been described in several organisms. The phenotypes of various allelic
combinations suggest that the strongest allele,
dd41, is not completely amorphic by genetic
criteria. Western blotting indicates that there is 80%-90% reduction
of the Dgrip91 protein in this allele. Although it is uncertain whether this residual protein has any function, this
and the genetic observations suggest there may be some residual
function of the gamma-TuRC even in the dd41 mutant (Barbosa, 2000).
It is generally thought that the late survival of larvae with extreme
mitotic defects reflects perdurance of maternal contribution to the
oocyte from the heterozygous mother, as has been shown for other
cell-cycle genes. In the case of dd4, this
assumption has been challenged by the report of normal oocytes from
homozygous dd41 mitotic ovarian clones arising in
heterozygous females. However, preliminary
observations of embryonic development in eggs produced from allelic
combinations weak enough to give viable escaper females
(dd4S/dd4S and
dd4S/dd43) do indicate that there
is a vital maternal contribution to the oocyte: such eggs appear to
have parental DNA but they fail to undergo any development. These
observations suggest that if mothers carrying weak enough allelic combinations to be compatible
with survival to adulthood cannot build a viable egg, then either the
observations of no maternal effect are in error, or the observed
clones have sufficient perdurance of the wild-type product to build eggs
indistinguishable from normal heterozygotes (Barbosa, 2000).
Together, Dgrip84, Dgrip91, and gamma-tubulin form the three major
components of the gamma-TuRC and are homologous to the budding yeast proteins Spc97, Spc98, and Tub4. Genetic and molecular studies show
interactions between these genes in budding yeast, and their requirement for SPB structure, duplication, and separation. Interactions between members of this
complex and other components of the SPB and spindle are only beginning
to be understood. Spc98, for example, binds to the N-terminal region of
Spc110p, a coiled-coil protein that spans the inner and central
plaques of the SPB. The calmodulin binding C terminus of this protein contacts the central plaque and the N-terminal region, the inner plaque. Thus Spc98 might
form an essential link between Spc110 and the spindle microtubules that
emanate from the inner plaque and the defective spindle structures seen
in spc98 mutants may be a direct consequence of defects in
this interaction. The phenotypes of spc98 mutants thus have
some parallels with dd4 mutants in abnormal spindle
microtubule density and organization, and it will be of interest to
determine whether Dgrip91 has similar interactions with specific
components of the centrosome (Barbosa, 2000).
The more drastic disruption of purified preparations of centrosomes
with the salt KI in vitro removes a set of proteins, including the gamma-TuRC,
CP60, CP190, CNN, and Asp, thus destroying their ability to organize
microtubules. The salt
treatment appears to leave behind unidentified core centrosomal
components, since the structure of the PCM is changed very little when
examined by electron microscopy. In contrast to salt extraction,
reduction of functional Dgrip91 has a differential effect upon the loss
of centrosomal antigens. CNN remains in distinct bodies at most of the
well focused poles, indicating that its centrosomal association is not
dependent upon the presence of the gamma-TuRC. The defects of
centrosomin(cnn) mutants have been characterized for
a number of alleles that show maternal effects and male sterility. These indicate that its function is required for the integrity of both
centrosomal and centriolar structures. Syncytial embryos derived from
centrosomin mutant mothers undertake up to 12 rounds of
mitosis upon spindles whose poles have no astral
microtubules and have very little or none of the
centrosomal proteins CP60, CP190, or gamma-tubulin. Together, these data imply that CNN appears to be more
important in holding the structure of the centrosome together than does
the gamma-TuRC, and this is perhaps to be expected from the predicted
coiled-coil nature of CNN (Barbosa, 2000).
It is clear that mitotic spindles can form and function in the absence
of centrosomes. Repeated rounds of mitosis are known to take place in
the absence of centrosomes in the unfertilized eggs of Sciara
flies. Moreover, in Drosophila eggs derived from polo mothers, the four products of
female meiosis are capable of undergoing many rounds of mitosis on
acentriolar spindles. These spindles strongly
resemble the meiotic spindles of female Drosophila in which
gamma-tubulin cannot be detected by immunostaining at these spindle
poles, even though it is apparently needed for spindle function. The ability to build a functional spindle in Xenopus extracts in the absence of centrosomes is also well documented and requires minus end directed motors such as dynein to
focus the poles. The consequences of removing
centrosomes from cells that have robust checkpoints to monitor spindle
assembly can vary, and could reflect either or both the cell line
studied and exactly how the experiment was performed. Microsurgical
removal of centrosomes has been reported to block future cycles of cell
division. However, laser directed ablation of either one or both centrosomes
does not prevent assembly of spindles that could successfully undertake
anaphase. The high mitotic index resulting from
partial disruption of the centrosome in dd4 mutants suggests a
mitotic delay likely to result from activation of the spindle integrity
checkpoint known to be functional in larval brain cells (Barbosa, 2000).
The distribution of the Asp following the apparent breakdown of the
gamma-TuRC gives insight into how these proteins might cooperate in
microtubule nucleation. It is known that, following KI depletion of
centrosomes, their ability to organize asters of microtubules can only
be restored by supplying a complementary cytoplasmic extract that
contains both the gamma-TuRC and functional Asp protein. In wild-type cells, Asp forms a hemispherical cup-like structure on the face of the spindle
microtubules suggesting that it is contacting the minus ends of these,
and not the astral microtubules. Astral microtubules are not seen in
dd4 mutant cells at either the light or EM levels, and the
spindle poles exhibit varying degrees of disorganization. Nevertheless,
the Asp protein is invariably present at the spindle poles even in
those extreme cases where individual bundles of microtubules are no
longer held together at a single poorly focused pole. In such cases Asp
appears at the very tips of these tubules as if it is providing some
capping property to their minus ends (Barbosa, 2000).
It is difficult to compare the effects of gamma-tub23C and
dd4 mutations upon the structure of the centrosome itself. Nevertheless, although the centrosome had
abnormal morphology judged by the distribution of CP190 (Bx63 antigen) in the gamma-tub23C mutant, the antigen was only noted as being
at pole-like structures. Unfortunately, there are currently no known mutants of the CP190 gene, and its function remains unknown.
CP190 exists in a complex with CP60, and both proteins are known to be
nuclear during interphase and move onto centrosomes at mitosis. The extent of interaction between
these proteins and gamma-tubulin is also unclear. Two complexes containing gamma-tubulin have been purified from Drosophila
embryos; the 3 MD gamma-TuRC itself, and a smaller complex of 240 kD that
appears to be a subunit of the larger one. The CP190-CP60 complex
does not appear to be present in either of these gamma-tubulin complexes
from which it was separable by gel-filtration. However, low levels of
gamma-tubulin can be detected in the eluate from immunoaffinity columns constructed from antibodies to CP190 and CP60. This has led to the speculation that
although these proteins may not assemble with each other in
stoichiometric ratios, they may still show interactions, either on an
affinity column in vitro or during centrosome assembly. Consistent with
this is the observation that following loss of the majority of the
gamma-TuRC from the centrosome in dd41, some CP190
remains in the centrosome, whereas some dissociates and clusters in
punctate arrays in the region of the spindle occupied by the condensed
chromosomes. In this sense CP190 may be obeying elements of a nuclear
localization signal that directs its interphase location, the nuclear
envelope undergoing incomplete breakdown during mitosis in
Drosophila to form a fenestrated envelope around the spindle (Barbosa, 2000).
Despite the differences in fixation procedures, several aspects of the
ultrastructure of the mitotic apparatus in dd4 cells as seen
by electron microscopy, such as the microtubule density, are concordant
with observations by immunofluorescence. Chromosomes are abnormally
condensed and the number and density of spindle microtubules is greatly
reduced in the mutant cells. The dispersion of the gamma-tubulin, which
is assumed to be the primary consequence of the dd4 mutations,
is reflected by disorganization of the PCM and altered centriole
morphology. Some gamma-tubulin has been shown to be localized
to the core of the centriole, and inactivation of
the gamma-tubulin gene in Paramecium leads to inhibition of the
duplication of the related structures, the basal bodies. The finding of fewer than four centrioles in the serial EM
sections of some dd4 mutant cells suggests a failure of
centriole duplication. However, the failure to find centrioles at one
of the poles in six dd4 mutant cells suggests that centrosome
separation is also dependent upon a functional gamma-TuRC. This may be
related to a function in correctly holding centrioles together, because
mother and daughter centrioles are rarely perpendicular. The extent to
which other centrosomal components found principally in the PCM can
contribute to structure of the centriole is not clear. Nonetheless, it
is interesting in this context that an isoform of CNN expressed during
spermatogenesis is localized both to the centrosomes and to the basal
body and has been shown by mutational analysis to be required for the
organization of the flagellar axoneme that develops from the spermatid
basal body (Barbosa, 2000).
During asymmetric cell division in the Drosophila nervous system, Numb segregates into one of two daughter cells where it is required for the establishment of the correct cell fate. Numb is uniformly cortical in interphase, but in late prophase, the protein concentrates in the cortical area overlying one of two centrosomes in an actin/myosin-dependent manner. What triggers the asymmetric localization of Numb at the onset of mitosis is unclear. The mitotic kinase Aurora-A is required for the asymmetric localization of Numb. In Drosophila sensory organ precursor (SOP) cells mutant for the aurora-A allele aurA37, Numb is uniformly localized around the cell cortex during mitosis and segregates into both daughter cells, leading to cell fate transformations in the SOP lineage. aurA37 mutant cells also fail to recruit Centrosomin (Cnn) and gamma-Tubulin to centrosomes during mitosis, leading to spindle morphology defects. However, Numb still localizes asymmetrically in cnn mutants or after disruption of microtubules, indicating that there are two independent functions for Aurora-A in centrosome maturation and asymmetric protein localization during mitosis. Using photobleaching of a GFP-Aurora fusion protein, it has been shown that two rapidly exchanging pools of Aurora-A are present in the cytoplasm and at the centrosome and might carry out these two functions. These results suggest that activation of the Aurora-A kinase at the onset of mitosis is required for the actin-dependent asymmetric localization of Numb. Aurora-A is also involved in centrosome maturation and spindle assembly, indicating that it regulates both actin- and microtubule-dependent processes in mitotic cells (Berdnik, 2002).
A mitotic function for Drosophila Aurora-A has been described before. In strong alleles of aurora-A, centrosomes fail to separate, leading to the generation of abnormal monopolar mitotic spindles, defects in chromosome segregation, and the formation of polyploid cells. aurA37 mutant cells, in contrast, complete mitosis and divide into two daughter cells and can therefore be used to characterize other aspects of Aurora-A function. To analyze mitotic spindles in aurA37 mutants, control and mutant pupae were stained for DNA and alpha-Tubulin. Bipolar mitotic spindles are formed in aurA37 mutants, but while microtubule minus ends converge on the centrosome in wild-type, they are less focused in aurA37 mutants. This spindle morphology phenotype could reflect a defect in centrosome function, and therefore aurA37 mutants were stained for the centrosomal marker gamma-Tubulin. In control SOP cells, gamma-Tubulin staining is weak during interphase, but two strong dots appear during mitosis, indicating that gamma-Tubulin is recruited to centrosomes. In 67% of the aurA37 mutant mitotic SOP cells, no strong dots of gamma-Tubulin staining were visible, indicating a failure to recruit the protein to centrosomes. This defect is not completely penetrant, since one dot was observed in 17% of the cells, and, in another 17%, two closely spaced dots were seen. Defects in mitotic recruitment of gamma-Tubulin have been described before in flies mutant for cnn, a centrosomal core component that is dispersed in interphase but localized to centrosomes during mitosis. aurA37 mutant pupae were therefore double stained for Cnn and gamma-Tubulin. In contrast to wild-type, where Cnn is detected in two strong dots at either spindle pole, the protein is undetectable on centrosomes of most aurA37 mutant SOP cells. Thus, Aurora-A is required for recruiting both gamma-Tubulin and Cnn to centrosomes during mitosis, and these defects in centrosome maturation might be the cause of the abnormal spindle morphology. Despite the spindle defects in aurA37 mutants, however, the two daughter cells of SOPs are still preferentially arranged along the anterior-posterior axis, indicating that spindle orientation is unaffected (Berdnik, 2002).
Cnn has been shown to be required for localization of gamma-Tubulin to centrosomes during mitosis. The defects in gamma-Tubulin localization in aurA37 mutants could therefore be an indirect consequence of the failure to recruit Cnn. To test whether the defects in Numb localization are also caused by the failure to recruit Cnn, cnn null mutant Drosophila pupae were stained for Numb, gamma-Tubulin, and DNA. Even though no Cnn protein could be detected, these flies are viable. As in wild-type, Numb localizes into a cortical crescent during late prophase in all cnn mutant SOP cells even though, in 9% of the mitotic SOP cells (n = 22), the Numb crescent is mispositioned and does not correlate with the orientation of the metaphase plate. Since gamma-Tubulin is not recruited to centrosomes in these mutants, it is concluded that neither Cnn nor gamma-Tubulin recruitment to mitotic centrosomes is required for the asymmetric localization of Numb (Berdnik, 2002).
The failure to localize Numb asymmetrically in aurA37 mutants could still be caused by the spindle defects. In neuroblasts, Numb localization still occurs after complete disruption of the mitotic spindle. To test the requirement of a mitotic spindle for Numb localization in SOP cells, wild-type pupae were incubated in 20 µg/ml colcemid for 1 or 2 hr and stained for Numb and gamma-Tubulin. After 1 hr of treatment, on average, nine SOP cells per pupal notum showed the mitotic arrest phenotype typical of microtubule inhibitors: chromosomes were no longer aligned in the metaphase plate, and centrosomes were distributed at random positions. Numb was still asymmetrically localized in 78% of these colcemid-arrested SOP cells. After 2 hr of treatment, the average number of metaphase-arrested SOP cells per notum increased to 27, and Numb was asymmetrically localized in 81% of them, indicating that new Numb crescents can be formed in the absence of a functional mitotic spindle. No effect on centrosome position was observed in a control experiment in which colcemid was omitted and Numb crescents were observed in 71% of the mitotic SOP cells. Thus, neither a functional mitotic spindle nor recruitment of Centrosomin and gamma-Tubulin to centrosomes are required for asymmetric localization of Numb. It is concluded that the defects in Numb localization observed in aurA37 mutants are not indirect consequences of the spindle or centrosome defects. Rather, they indicate an independent role for Aurora-A in asymmetric protein localization during mitosis (Berdnik, 2002).
The γ-tubulin ring complex (γTuRC), consisting
of multiple protein subunits, can nucleate microtubule assembly.
Although many subunits of the γTuRC have been identified, a complete
set remains to be defined in any organism. In addition, how the
subunits interact with each other to assemble into γTuRC remains
largely unknown. This study reports the characterization of a novel
γTuRC subunit, Drosophila gamma ring protein with WD
repeats (Dgp71WD). With the exception of γ-tubulin, Dgp71WD is the
only γTuRC component identified to date that does not contain the
grip motifs, which are signature sequences conserved in γTuRC
components. By performing immunoprecipitations after pair-wise
coexpression in Sf9 cells, it was shown that Dgp71WD directly interacts with
the grip motif-containing γTuRC subunits, Dgrips84, 91, 128, and
163, suggesting that Dgp71WD may play a scaffolding role in γTuRC
organization. Dgrips128 and 163, like Dgrips84 and
91, can interact directly with γ-tubulin. Coexpression of any of
these grip motif-containing proteins with γ-tubulin promotes
γ-tubulin binding to guanine nucleotide. In contrast, in the same
assay Dgp71WD interacts with γ-tubulin but does not facilitate
nucleotide binding (Gunawardane, 2003).
The Drosophila γTuRC consists of ~8 polypeptides of
which γ-tubulin and Dgrips75 (76p), 84, 91, 128, and 163 have been identified and characterized. Interestingly, all of these Dgrips contain conserved grip motifs. On the basis of the protein profile of the purified
Drosophila γTuRC, it is estimated that two to three
additional Dgrips with apparent molecular masses of ~75 kDa remain
to be cloned and characterized. Through database searches,
it was found that several peptide sequences that were obtained by
microsequencing of the purified γTuRC matched a Drosophila
partial EST. The full-length cDNA was cloned and found to contained five WD repeats. Therefore, this novel protein is referred to as Dgp71WD, which stands for
Drosophila gamma ring protein of 71 kDa with WD repeats (Dgp71WD,
accession number AF461267). Interestingly, Dgp71WD did not contain the
grip motifs found in all of the other Dgrips identified thus far.
Database searches revealed a large number of proteins from various
organisms sharing sequence homology with the WD-repeat regions of
Dgp71WD. The alignment the five WD repeats in Dgp71WD align with the WD
repeats of the Arabidopsis photomorphogenesis repressor COP1 and the mouse
Nedd1. Interestingly, the presently uncharacterized mouse Nedd1
is similar in size to Dgp71WD and both proteins have the WD repeats at
their N-termini. Pair-wise alignment showed that the
overall amino acid identity between Nedd1 and Dgp71WD is 20.8% and
that the homology between the two proteins extends beyond the WD
repeats (Gunawardane, 2003).
To determine if Dgp71WD is a γTuRC subunit, rabbit polyclonal
antibodies against the C-terminal peptide of γ-tubulin (DrosC) or against a peptide corresponding to amino acids 399-417 of Dgp71WD were used to immunoprecipitate Dgp71WD from Drosophila embryo extract. Anti-Dgp71WD
antibody was found to immunoprecipitate the same set of γTuRC subunits as does the anti-γ-tubulin antibody, suggesting that Dgp71WD is a subunit of
the γTuRC. Furthermore, the antibody against Dgp71WD recognizes a
protein of ~71 kDa in γTuRC immunoprecipitated with γ-tubulin
antibodies. In addition, when Drosophila embryo extracts are subjected to
sucrose gradient sedimentation, Dgp71WD comigrate with the other
γTuRC subunits. Because the previously characterized
γTuRC subunits localize to the centrosome throughout the cell cycle,
whether Dgp71WD shows a similar localization pattern was tested. Dgp71WD was found to colocalize with γ-tubulin at centrosomes in
Drosophila embryos during interphase and mitosis. Taken together, these results showed that Dgp71WD is a subunit of the Drosophila γTuRC (Gunawardane, 2003).
Because Dgp71WD contains WD repeats and no grip motifs, it was
reasoned that it might play a scaffolding role by binding to the grip
motif-containing subunits. If this is true, Dgrips84, 91, 128, and 163 should directly interact with Dgp71WD. To test this possibility, Dgp71WD was coexpressed with each of the Flag-tagged Dgrips84, 91, 163, or untagged Dgrip128, in Sf9 cells. Specific antibodies that recognize Dgp71WD, Dgrips84, 91, 128, 163, or Flag antibody were used to immunoprecipitate each of the proteins. The
immunoprecipitates were first separated by SDS-PAGE and the proteins
were either stained by Coomassie Blue or probed by Western blotting
with specific antibodies. It was found that the Dgp71WD antibody
immunoprecipitates Dgrips84, 91, 128, and 163 . Conversely, antibodies that immunoprecipitate each of the Dgrips also immunoprecipitate Dgp71WD. On the basis of these results, it was concluded that
Dgp71WD interacts with each of the four grip-motif-containing γTuRC
subunits (Gunawardane, 2003).
Next, it was asked whether Dgp71WD also directly interacts with
γ-tubulin. Dgp71WD and γ-tubulin were coexpressed in Sf9 cells and antibodies against each of the proteins were used for immunoprecipitation. It was found that although the antibody against γ-tubulin
immunoprecipitates Dgp71WD, the antibody against Dgp71WD does not
immunoprecipitate γ-tubulin. The lack of reciprocal immunoprecipitation could reflect a lack of interaction between γ-tubulin and Dgp71WD. Alternatively, it
is possible that Dgp71WD and γ-tubulin do interact with each other,
but the binding of this particular antibody to Dgp71WD disrupts this interaction. The disruption is possible if the Dgp71WD antibody bind to the region of Dgp71WD involved in its binding to γ-tubulin. In contrast, DrosC
and Dγ2 antibodies do immunoprecipitate Dgp71WD. On the basis of these results, it is concluded that Dgp71WD and γ-tubulin directly interact with each other (Gunawardane, 2003).
Attempts were made to develop an assay to compare the functional
consequences of the binding of Dgp71WD to γ-tubulin with that of the
grip motif-containing proteins. Because γ-tubulin in γTuRC and
γTuSC can bind to guanine nucleotides, it was reasoned that the
nucleotide binding of γ-tubulin could be dependent on its interaction
with other γTuRC subunits. Therefore, it was asked whether γ-tubulin
expressed alone or together with Dgrip84 or Dgrip91 could bind to GTP.
Using a UV cross-linking assay, it was found that γ-tubulin when expressed alone and purified, does not bind to GTP. However, when γ-tubulin is coexpressed with either Dgrip84 or
Dgrip91 in Sf9 cells and purified, it is able to bind to GTP in the
cross-linking assay. As expected, γ-tubulin in the
baculovirus reconstituted γTuSC also binds to GTP. These findings
suggest that the interactions between the Dgrip84 and Dgrip91 with
γ-tubulin facilitate γ-tubulin binding to GTP (Gunawardane, 2003).
These finding prompted a test of whether Dgp71WD, Dgrip128, and
Dgrip163 could also facilitate γ-tubulin binding to GTP. γ-Tubulin
was coexpressed with Dgp71WD, Dgrip128, or Dgrip163, isolated, and
assayed for GTP binding using the UV cross-linking assay as above. It was found
that γ-tubulin binds to GTP when coexpressed with either Dgrip128 or
Dgrip163, but not when coexpressed with Dgp71WD. Furthermore, γ-tubulin
binds to GTP when coexpressed with all three subunits,
suggesting that Dgp71WD does not inhibit GTP binding. These studies
suggest that the interaction between γ-tubulin and the grip
motif-containing subunits facilitate γ-tubulin binding to GTP,
but the interaction between γ-tubulin and Dgp71WD does not. From these
studies, it is concluded that the nature of the interaction of Dgp71WD with
γ-tubulin differs from that of the grip motif-containing γTuRC
subunits (Gunawardane, 2003).
The finding that all Dgrips interact with γ-tubulin directly is
intriguing because it suggests that the Dgrips75, 79, 128, and 163 that
were originally thought to be the cap subunits, directly contact
γ-tubulin in the γTuRC. If this is the case, the current γTuRC
model, which hypothesizes that the γTuRC ring consists exclusively of
γTuSCs, would need to be revised. It is speculated that each of the
Dgrips75, 79, 128, and 163 can form dimers with γ-tubulin molecules. These dimers along with several γTuSCs may be required to
form the ring of the γTuRC (Gunawardane, 2003).
Structural studies revealed that WD repeats form a ß-propeller
fold that mediate protein-protein interactions. This study suggests that Dgp71WD could provide a scaffold via
its WD-repeats to tether all of the Dgrips together in the γTuRC.
Consistent with this idea, it was found that four Dgrips (84, 91, 128, and
163) interact directly with Dgp71WD. Clearly, further structural and
biochemical studies are necessary to determine whether some or all of
the Dgrips75, 79, 128, and 163 participate in the formation of the
γTuRC ring. Also, it is important to determine whether and how any of
these Dgrips participate in the formation of the cap structure of the
γTuRC (Gunawardane, 2003).
Coexpressing any one of the grip motif-containing
subunits, Dgrips84, 91, 128, and 163, with γ-tubulin is sufficient to promote γ-tubulin to bind to GTP. However, coexpressing
γ-tubulin with Dgp71WD, which does not contain grip motifs, does not
facilitate γ-tubulin binding to GTP. This finding shows that the
interactions between γ-tubulin and Dgrips have a significantly
different consequence from the interaction between γ-tubulin and
Dgp71WD. Furthermore, it suggests that the grip motif-containing
subunits play a role in regulating the GTP binding properties of
γ-tubulin. Because GTP is important for alpha- and ß-tubulin
function, it is suspected that Dgrips may be important for γ-tubulin
function. Consistent with this idea, it was found that γ-tubulin
expressed alone does not incorporate into γTuRC in vitro; but
coexpressing one of the Dgrips with γ-tubulin is sufficient for the
incorporation (Gunawardane, 2003).
Previously, human and Chlamydomonas γ-tubulins expressed
alone using rabbit reticulocyte lysate and baculovirus expression systems, respectively, were used to study the microtubule binding and
nucleating activities of γ-tubulin. However,
whether the γ-tubulins could bind to guanine nucleotides was not
determined. Because it was found that γ-tubulin expressed
alone does not bind GTP, caution should be used in analyzing the
function of γ-tubulin in the absence of Dgrips (Gunawardane, 2003).
The interaction observed between Dgp71WD and γ-tubulin should also
be interpreted cautiously. Because γ-tubulin found in γTuRC and
γTuSC can be cross-linked to GTP, the inability of γ-tubulin to
bind to GTP when coexpressed with Dgp71WD may indicate that γ-tubulin
does not assume the same structural conformation as in the γTuRC. One
possibility is that the γ-tubulin coexpressed with Dgp71WD is not
folded properly. If so, the interaction between Dgp71WD and γ-tubulin
that observed may not reflect a true interaction in the γTuRC.
Alternatively, it is possible that the γ-tubulin expressed with
Dgp71WD is folded correctly, but assumes a slightly different
conformation to disfavor GTP binding in the in vitro assays. If so, the
interaction between Dgp71WD and γ-tubulin is likely to reflect a true
interaction in the γTuRC. Structural studies of γTuRC will be
important to resolve whether γ-tubulin directly contact Dgp71WD in
the complex (Gunawardane, 2003).
A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. This study shows that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, Centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN (Terada, 2003).
In animal cells, microtubules are organized from the centrosome/microtubule-organizing center (MTOC), composed of a pair of centrioles and the surrounding pericentriolar material. Individual microtubules are nucleated from an ~25-nm γ-tubulin–containing ring complex (γ-TuRC). At the onset of M phase, the centrosome becomes 'mature' and organizes more microtubules, which is accompanied with an increased level of γ-tubulin accumulation at each spindle pole. One of the molecules that has been implicated in the mechanism of centrosome maturation is Aurora-A, a mitosis-specific Ser/Thr kinase located at mitotic poles and spindle microtubules. The kinase, originally identified as a gene product important in spindle assembly and function in Drosophila, has recently been shown to be in the Ran-signaling pathway and to play an important role in efficient transmission of Ran-GTP gradient established by the condensed chromosomes for the control of spindle assembly and dynamics. Aurora-A binds to spindle components, such as TACC/XMAP215 and TPX2. Although possible functions of those molecules and their interaction with Aurora-A in bipolar spindle formation have been elucidated, mechanisms of how Aurora-A stimulates the recruitment of γ-tubulin to the centrosome at spindle poles have not yet been evaluated. To address this question, centrosomal proteins were sought that interact with Aurora-A and regulate the process of microtubule nucleation onto the centrosome (Terada, 2003).
By screening of a Drosophila two-hybrid library, two clones were isolated encoding a molecule capable of interaction with Aurora-A. The sequence corresponds to the COOH-terminal domain of centrosomin (CNN), a core component of the centrosome important for assembly of mitotic centrosomes in Drosophila. Although the truncated polypeptide covered by clone CNN-C1 appears to be sufficient for interaction with Aurora-A, the binding intensity was weaker than CNN-C. Endogenous Aurora-A, but not Aurora-B, immunoprecipitates with HA-tagged CNN expressed in S2 cells. Specificity of the COOH-terminal domain of CNN for interaction with Aurora-A was further confirmed by in vitro binding assays (Terada, 2003).
To investigate the role of protein interaction in the centrosome, S2 cells were prepared from which Aurora-A or CNN was depleted by RNA interference (RNAi). In cells lacking Aurora-A, not only CNN, but also γ-tubulin, were absent at each spindle pole. When CNN was depleted, neither γ-tubulin nor Aurora-A was seen at the spindle pole. In cells with partially depleted Aurora-A or CNN, comparable amounts of γ-tubulin and CNN or Aurora-A were detected at each pole. Besides γ-tubulin, other centrosome proteins, CP190 (a–c) and CP60 (d–f), become dislocated from the spindle poles in RNAi cells. Therefore, it is concluded that CNN and Aurora-A are mutually dependent for localization at spindle poles, which is required for proper targeting of other centrosomal proteins to the centrosome. This is consistent with previous observations (Barbosa, 2000) that the centrosomal association of CNN is not dependent on the presence of γ-tubulin/γ-TuRC (Terada, 2003).
To confirm the role of CNN in recruiting γ-tubulin, protein interaction was analyzed in vitro. Nickel beads conjugated with His-tagged CNN were mixed with cell extracts prepared from colcemid-treated S2 cells. γ-Tubulin was specifically sedimented by the full and NH2-terminal sequence, but not the COOH-terminal sequence of CNN. Because neither in vitro binding assays nor two-hybrid screens demonstrated direct binding between two molecules, CNN may interact with a γ-tubulin complex, rather than γ-tubulin directly. Further, HA-tagged CNN was expressed in S2 cells. Exogenous proteins caused formation of γ-tubulin–containing cytoplasmic aggregates capable of microtubule formation and association with microtubule asters. These results clearly indicate that the NH2-terminal domain of CNN interacts with γ-tubulin/γ-TuRC and plays an important role in assembly of MTOCs (Terada, 2003).
γ-Tubulin/γ-TuRC–mediated microtubule assembly is believed to be common among species. Thus, it is highly likely that an Aurora-A–binding molecule(s) equivalent to CNN is functioning in a variety of organisms. Although Drosophila CNN was unable to associate with mammalian Aurora-A in transfected mammalian cells as well as by two-hybrid screens, the NH2-terminal domain of CNN still interacts with γ-tubulin/γ-TuRC in mammalian cells as in S2 cells. To analyze a possible role of CNN–γ-tubulin interaction in initiation of microtubule assembly, Drosophila CNN was overexpressed in mammalian cells. HA-tagged CNN induces cytoplasmic foci in various sizes and numbers. Significantly, the pattern of microtubule distribution is profoundly affected as a result of microtubule association with virtually every dot containing CNN. These sites can initiate microtubule formation as evidently shown in cells where short microtubules are assembled during brief recovery from nocodazole treatment. All cells overexpressing CNN induced microtubule-organizing sites, which were associated with centrosome proteins, such as pericentrin and Cep135. Particularly prominent was γ-tubulin, which was probably recruited from a large cytoplasmic pool. In support of this view, GFP-tagged exogenous γ-tubulin became colocalized with HA-CNN to participate in the formation of microtubule-nucleating sites. This was in striking contrast with cells expressing γ-tubulin alone, where cytoplasmic aggregates induced by γ-tubulin expression could not contribute to microtubule formation. These results suggest that microtubules are directly nucleated from the CNN aggregates through the mechanism mediated by γ-tubulin/γ-TuRC (Terada, 2003).
To confirm the microtubule-nucleating activity of the CNN aggregates, microtubules were polymerized in vitro by incubating isolated GFP-tagged CNN dots with X-rhodamine–conjugated brain tubulin. There was always a dot positive in GFP fluorescence at the center of the microtubule asters. Although variable numbers of microtubules emanated from the center, more microtubules tended to polymerize onto the GFP dots in larger sizes. The process of aster formation was monitored by time-lapse microscopy. A fluorescence image taken 10 min after mounting the sample on a microscopic stage revealed several microtubules growing from a GFP-positive site. As time progressed, more microtubules appeared to emanate from the center, indicating that microtubules were formed by direct polymerization onto the CNN-containing foci, rather than that preformed microtubules were gathered around the center (Terada, 2003).
Microtubules are nucleated from the pericentriolar material that surrounds the centrioles of the centrosome. To compare ultrastructure of microtubule-initiating sites induced by CNN with that of the pericentriolar material/centrosome, CHO cells expressing GFP-tagged CNN were examined by EM. Two microtubule asters were seen formed in cells that were briefly extracted before fixation. Located at each focal point of microtubule asters was an electron-dense particle in various sizes and shapes. Unlike the pericentriolar material, which has been described as an ill-defined amorphous cloud, the entire structure induced by CNN was well delineated by electron-dense materials to which microtubules were attached. In favorable sections, microtubules could be seen penetrating to the interior region of the aggregates. Neither centrioles nor centrosomal substructures, such as satellites, appendages, and CHO cell–specific virus particles, were generally seen at the site induced by CNN expression. Because CNN is a coiled-coil structural protein (Heuer, 1995), the dense particles likely represent the aggregated form of overexpressed CNN proteins (Terada, 2003).
Multiple centrosomes/MTOCs have been detected in cells in which the mechanism of centrosome duplication coupled with the cell cycle control becomes deregulated. In the case of CNN-containing MTOCs, their number and size formed during relatively short periods (8–12 h) varied greatly according to the level of protein expression. Moreover, no centrioles were found at ectopic MTOCs by EM and immunostaining with centriole-specific centrin-2 antibodies. Therefore, it is plausible that CNN expression causes the formation of protein aggregates that acquire the microtubule-nucleating capacity by recruiting γ-tubulin/γ-TuRC. This unique property of CNN to generate microtubule-nucleating sites by interacting with γ-tubulin/γ-TuRC suggested CNN may function as an adaptor for connecting γ-tubulin to the centrosome (Terada, 2003).
By expressing truncated polypeptides, it was concluded that CNN's ability to interact with γ-tubulin/γ-TuRC and induce ectopic microtubule-nucleating sites resides in the NH2-terminal sequence of CNN from which the Aurora-A–binding domain is omitted. In contrast, cytoplasmic aggregates formed in cells expressing the COOH-terminal domain failed to initiate microtubule formation in both S2 and mammalian cells. These results lead to the conclusion that CNN consists of two functionally distinct subdomains: the Aurora-A–binding site is at the COOH terminus capable of formation of the protein complex to be recruited to the spindle pole, and the NH2-terminal sequence is involved in assembling centrosomes/MTOCs by recruiting γ-tubulin/γ-TuRC. Although no CNN homologues have yet been identified outside Drosophila, Aurora-A would likely be involved in the control of microtubule nucleation through its association with the COOH terminus of a CNN-related molecule(s) in mammalian cells (Terada, 2003).
Control of mitotic spindle assembly onto the