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First published online 13 September 2006
doi: 10.1242/dev.02570
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TuRC components Grip75 and Grip128 have an essential microtubule-anchoring function in the Drosophila germline
1 Max-Planck-Institute for Developmental Biology, Department of Genetics,
Spemannstr. 35, 72076 Tübingen, Germany.
2 Max-Planck-Institute for Developmental Biology, Electron Microscopy Unit,
Spemannstr. 35, 72076 Tübingen, Germany.
* Author for correspondence (e-mail: nina.vogt{at}tuebingen.mpg.de)
Accepted 7 August 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-tubulin ring complex (TuRC) forms an essential template
for microtubule nucleation in animal cells. The molecular composition of the
TuRC 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 TuRC 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. We report 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.
We propose that Grip75 and Grip128 are required to tether microtubules at
specific microtubule-organizing centers, instead of being required for general
microtubule nucleation. The TuRC cap structure may be essential only
for non-centrosome-based microtubule functions.
Key words: -Tubulin ring complex, Drosophila, bicoid RNA localization, Meiosis, Spermatogenesis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-Tubulin is essential for microtubule nucleation in vivo
(Wiese and Zheng, 1999
). Two
-tubulin containing complexes, the -tubulin small complex
(TuSC) and the -tubulin ring complex (TuRC), have been
isolated from a variety of sources (Murphy
et al., 1998; Oegema et al.,
1999; Zheng et al.,
1995). The Drosophila TuSC, which contains
-tubulin, Grip91 and Grip84, displays low microtubule-nucleating
activity in vitro (Oegema et al.,
1999). The larger TuRC consists of a lockwasher-like
structure and a globular cap that decorates one end of the complex
(Moritz et al., 2000). It
contains several TuSCs and Grip71, Grip75, Grip128 and Grip163
(Gunawardane et al., 2000;
Gunawardane et al., 2003;
Oegema et al., 1999). The
TuSC has been suggested to form the subunits of the lockwasher, whereas
the remaining Grip proteins may build the cap
(Moritz et al., 2000;
Zhang et al., 2000). The
TuRC is associated with microtubule minus ends, possesses high
microtubule-nucleating activity in vitro and forms a template for microtubule
nucleation in vivo (Moritz et al.,
2000; Zheng et al.,
1995).
The structural organization of the TuRC into the lockwasher and the
cap may reflect a functional subdivision. The components of the TuSC
appear to be required for microtubule organization. In Drosophila,
mutations in Grip91 (l(1)dd4-FlyBase) or Grip84 are
lethal and display defects in spindle assembly
(Barbosa et al., 2000;
Colombie et al., 2006).
Drosophila has two -tubulin genes,
Tub23C and Tub37C. Whereas Tub37C
expression is restricted to the female germline and the early embryo,
Tub23C is almost ubiquitously expressed and crucial for mitosis
(Sunkel et al., 1995;
Tavosanis et al., 1997). In
contrast to the TuSC components, the function of the cap components is
poorly understood. Although in vitro data suggest that only the TuRC
but not the TuSC provides high microtubule-nucleating activity, null
mutations in the TuRC component Grip75 are viable
(Schnorrer et al., 2002).
Furthermore, depletion of cap components by RNAi in S2 cells results in mild
mitotic defects (Verollet et al.,
2006). These data suggest that either the TuSC can nucleate
microtubules in vivo to an extent that is sufficient for life, or that Grip75
is dispensable for TuRC function in microtubule nucleation.
Tub37C and Grip75 are essential for the microtubule-dependent
localization of bicoid (bcd) RNA to the anterior cortex of
the Drosophila oocyte (Schnorrer
et al., 2002). In the oocyte, bcd RNA initially localizes
in a ring at the anterior cortex. At stage 10b, a transition into a disc-like
localization pattern occurs. bcd RNA remains at the anterior cortex
until the egg is laid (St Johnston,
2005). In Grip75 and Tub37C mutant
oocytes, relocalization during stage 10b fails and bcd RNA diffuses
away from the anterior cortex (Schnorrer
et al., 2002). Grip75 and Tub37C are concentrated together
with bcd RNA at the anterior cortex at this stage, and, thus, it has
been proposed that a new microtubule-organizing center (MTOC) assembles at the
anterior cortex at stage 10b.
Are Grip71, Grip128 and Grip163 required for the same processes as Grip75,
or do the individual subunits have different functions from Grip75? As
Grip71, Grip128 or Grip163 mutants were not available, it
was unclear if the Grip75 mutant phenotype resembles a `cap-null'
situation, and why the cap structure of the TuRC was not essential for
the microtubule-nucleating activity of the TuRC.
We have isolated mutants in Grip128, which mislocalize
bcd RNA during late oogenesis in the same way as
Tub37C and Grip75 mutants. Grip75 and
Grip128 mutants are viable but display defects in male and female
meiosis, as well as in sperm motility. We provide evidence that a TuRC
forms in Grip128 and Grip75 mutants, suggesting that the
TuRC is functional in microtubule nucleation without the full cap
structure. However, specific functions of the TuRC require the
additional proteins Grip128 and Grip75. We propose that Grip128 and Grip75
anchor the TuRC at special MTOCs, rather than being essential for
microtubule nucleation.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
Grip75175 and Tub37C139
(Schnorrer et al., 2002),
Grip128326 and Grip128352 (this
study), and NZ143.2 (Clark et al.,
1997). Transgenic flies were generated in a y w
background according to standard methods. Germline clones were induced by
incubating third instar larvae grown in vials once for 1 hour or in bottles
twice for 2 hours on 2 consecutive days in a 37°C water bath. Homozygous
mutant Grip128 females were generated by rescuing the sterility of
Grip128 mutant males with the genomic rescue construct and
backcrossing to Grip128 heterozygous females.
Screening procedure
Grip128 mutants were isolated in the course of an F1 screen
designed to identify new mutants that disrupt bcd RNA localization
during oogenesis. Screening was carried out as described
(Luschnig et al., 2004;
Schnorrer et al., 2002) with
the modification that we established lines from females that produced eggs
with early arrest phenotypes, but also with other strong developmental
defects. Mutations were induced on a w f hs-Flp122 FRT9-2
chromosome.
Identification of Grip128 mutants
The mutation X-326 was mapped between 14,650 kb and 15,591 kb on
the physical map (release 3.2) using a combination of conventional and
SNP-based meiotic mapping (Berger et al.,
2001). Details are available upon request. We PCR-amplified
suitable fragments from genomic DNA and sequenced the candidate gene
Grip128 from X-326, X-352 and the parental chromosome.
Molecular biology
For the genomic rescue construct, Grip128 was amplified from
genomic DNA and cloned into pCaSpeR4 using a XbaI compatible
NheI site about 1.4 kb upstream of the start codon and a
XhoI site (underlined) introduced with primer
aactcgagtgaggagttcgagtgaggttttg.
Protein biochemistry
Preparation of ovarian extracts, protein expression analysis and
immunoprecipitations were performed as described
(Schnorrer et al., 2002). We
used extract buffer [50 mM HEPES-KOH pH 7.6, 75 mM KCl, 1 mM EGTA, 1 mM EDTA,
0.05% NP-40, 1 mM DTT, 1 mM PMSF, protease inhibitor mix (Roche)] for all
experiments. Sucrose density gradients were prepared as described
(Moritz et al., 1998), with
the following modifications. Ovarian extract (100 µl) was loaded onto each
5-40% sucrose gradient (50 mM HEPES-KOH pH 7.6, 75 mM KCl, 1 mM
MgCl2, 1 mM EGTA, 1 mM DTT), and the gradients were centrifuged at
237,000 g in a SW60 rotor (Beckman) for 4 hours at 4°C.
The ribosomal profile was measured at 260 nm, and the peak of the small
ribosomal subunit was used as a 40 S size standard. Homozygous mutant tissue
was used for all biochemical assays. For detection, we used the following
primary antibodies: rabbit anti-Grip128 (1:5000) and anti-Grip163 (1:5000)
(Gunawardane et al., 2000);
rabbit anti-Grip91 (1:2000), anti--tubulinC12 (1:5000) and anti-Grip84
(1:2000) (Oegema et al.,
1999); rabbit anti-Grip75 affinity-purified (1:2000)
(Schnorrer et al., 2002);
rabbit anti-Swa serum (1:20000) (Schnorrer
et al., 2000); and mouse anti--tubulin GTU88 (1:5000)
(Sigma). Both -tubulin antibodies show the same specificity on western
blots. Primary antibodies were detected with goat anti-mouse-HRP (1:5000)
(Dianova), donkey antirabbit-HRP (1:10000) (Amersham) or ProteinA-HRP (1:5000)
(Amersham) followed by enhanced chemiluminescence.
Cytology
In situ hybridization and analysis of cytoplasmic streaming were performed
as described (Schnorrer et al.,
2002). Stage 14 oocytes were fixed as described
(Tavosanis et al., 1997) and
stained with 1 µg/ml DAPI.
For microtubule staining in oocytes, ovaries were dissected and fixed in
methanol, rehydrated into PBT (PBS containing 0.1% Tween-20) and blocked in 5%
normal goat serum (NGS). Incubation with FITC-conjugated anti-
-tubulin
DM1A (Sigma) at 1:100 was overnight at 4°C. Ovaries were then washed in
PBT, dehydrated in methanol and embedded in a 2:1 mixture of benzyl benzoate
and benzyl alcohol.
For other oocyte staining, ovaries were fixed in 4% paraformaldehyde in PBT, washed in PBTx (PBS containing 0.2% Triton-X-100) and blocked in 5% NGS. Incubation with primary antibody was overnight at 4°C. Ovaries were then washed in PBTx, incubated with the secondary antibody for at least two hours, washed and embedded in Aqua/Polymount (Polysciences).
Zero- to 30-minute-old embryos were fixed in methanol/heptane, washed in methanol, rehydrated into PBT and further processed as above. Embryos were embedded in Aqua/Polymount or benzyl benzoate/benzyl alcohol.
Testes were dissected in testes buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris-HCl pH 6.8, 1 mM EDTA) and squashed on SuperFrost slides. The slides were frozen in liquid nitrogen, the coverslip was removed and the testes fixed in cold methanol. Testes were rehydrated into PBT and blocked in 5% NGS. Incubation with primary antibody containing 20 µg/ml RNAse A was for 1 hour at room temperature. Testes were then washed in PBT and incubated with the secondary antibody and 25 µg/ml propidium iodide for one hour. After washing, the testes were embedded in Aqua/Polymount.
We used the following antibodies: mouse anti-ß-Gal (1:2000) (Promega),
anti--tubulin GTU88 (1:100) and anti--tubulin DM1A (1:1000)
(Sigma), and goat anti-mouse-Al488 (Molecular Probes) (1:500). Images were
collected on a confocal microscope (Zeiss LSM510).
Electron microscopy
Testes were prepared for electron microscopy with the DMSO-trialdehyde
fixation method (Kalt and Tandler,
1971). Briefly, testes were incubated in fixative (100 mM sodium
cacodylate, 3% glutaraldehyde, 2% formaldehyde, 1% acrolein, 2.5% DMSO) for 30
minutes at room temperature and then kept on ice for further 3 hours. Samples
were postfixed on ice with 1% osmium tetroxide in 100 mM phosphate buffer and
then embedded in 2% agarose. The testes were contrasted with 1% tannic acid
and then 1% uranyl acetate in water, dehydrated with ethanol, embedded in epon
and sectioned for transmission electron microscopy. Images were acquired with
a Philips CM10 transmission electron microscope at 60 kV.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In an F1 screen designed to identify lethal and/or early
embryonic arrest mutants with defects in bcd RNA localization,
mutants in Grip75 and Tub37C were identified
(Luschnig et al., 2004;
Schnorrer et al., 2002).
To identify additional mutants that disrupt bcd RNA localization,
we extended the F1 screen to the X chromosome. In brief, males were
mutagenized and crossed to females containing a GFP marker on the X
chromosome. Using the Flp-FRT system, clones were induced in the germline by
mitotic recombination. Eggs derived from homozygous mutant germline clones
were identified by the absence of GFP fluorescence. When eggs showed defects
in their development, lines were established from sibling eggs derived from
heterozygous germline clones. These resulting lines were again tested for
their phenotype using the Flp-FRT/DFS system that eliminated all but
homozygous mutant clones (Chou and
Perrimon, 1992; Chou and
Perrimon, 1996), and upon confirmation, ovaries were assayed for
bcd RNA localization.
Nine and a half thousand females produced non-fluorescent progeny that were scored for developmental defects. 174 lines were established and screened by in situ hybridization. In this paper, we describe two mutant alleles of the same gene, X-326 and X-352, with a specific defect in bcd RNA localization at stage 10b. Both X-326 and X-352 mutants are viable but male and female sterile. Eggs derived from homozygous mutant germline clones do not undergo nuclear divisions, as judged by DIC microscopy of embryos under oil (data not shown).
Identification of Grip128 mutants
To identify the gene disrupted in the X-326 and X-352
mutants, we mapped X-326 by meiotic recombination between the visible
markers garnet and forked. To refine our mapping, we used
single nucleotide polymorphisms and mapped the mutation between 14,650 kb and
15,591 kb on the physical map (release 3.2). A candidate gene in this interval
was Grip128. Sequencing this gene revealed nonsense mutations in the
X-326 mutant (Gln662
stop) and in the X-352
mutant (Gln706stop) (Fig.
1A).
To support these data, we generated a 5.2 kb genomic rescue construct encompassing 1.4 kb of upstream sequences, the full coding region, the introns and the predicted 3' untranslated region of the Grip128 gene. The corresponding transgene fully rescued the male and female sterility of both mutants. Therefore, we conclude that the mutations causing the sterility in the X-326 and X-352 mutants are in the Grip128 gene and we named the two mutants Grip128326 and Grip128352.
To confirm the predicted truncations, we analyzed Grip128 expression in
wild-type and mutant ovarian and male extracts. An antibody specific to
Grip128 recognized a protein at the expected size of 
130 kDa in wild-type
ovaries and males (Fig. 1B)
(Gunawardane et al., 2000).
However, we could not detect any protein of the wild-type or predicted
truncated sizes of 76 kDa and 81 kDa in extracts of
Grip128326 and Grip128352 mutant
ovaries or males, even though the available antibody was generated against the
first 200 amino acids of Grip128
(Gunawardane et al., 2000).
The additional bands detected by the antibody are not specific, as an antibody
we raised against amino acids 192-479 shows a different background pattern
(data not shown). Grip128326 over the deficiency
In(1)AC2[L]AB[R], which uncovers Grip128, is viable and shows the
same bcd mislocalization phenotype as Grip128326
or Grip128352 homozygotes (data not shown). We therefore
conclude that both alleles are protein-null alleles and also behave
genetically as null alleles.
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Tub37C are required for bcd RNA relocalization
in stage 10b (Schnorrer et al.,
2002). To determine whether Grip128 has a similar function, we
analyzed bcd RNA distribution in Grip128 mutant oocytes and
eggs. In wild-type and Grip128 mutant oocytes, bcd RNA is
localized in a ring at the anterior cortex prior to stage 10b
(Fig. 2A,D). However, the
transition into the disc-like pattern only partially occurs and bcd
RNA then diffuses away from the anterior cortex in Grip128 mutants
(Fig. 2E). bcd RNA is
completely unlocalized in mutant stage 12-13 oocytes (data not shown), whereas
in Grip128 mutant eggs, bcd RNA is distributed in a graded
manner (Fig. 2F), which is
probably due to the bcd RNA destabilizing activity of the posterior
system. Hence, the defect in bcd RNA localization in Grip128
mutants is identical to the defects of Grip75 and
Tub37C mutants, suggesting a similar role in the bcd
RNA localization machinery. Moreover, the bcd mislocalization
phenotype of Grip128352;Grip75175 double mutant
oocytes is the same as in the single mutants (data not shown), without obvious
differences in the strength of the phenotype.
Grip75 and Tub37C are enriched at the anterior cortex of stage 10b
and 11 oocytes, and participate in a new MTOC, which directs the
relocalization of bcd RNA
(Schnorrer et al., 2002). To
demonstrate a functional requirement of Grip128 in this MTOC, we analyzed
Nod:ßgal and microtubule distribution in wild-type and Grip128
mutant oocytes. Nod:ßgal is a marker for microtubule minus-ends, which
recapitulates bcd RNA localization in wild-type oocytes
(Clark et al., 1997;
Schnorrer et al., 2002). In
Grip128 mutant oocytes, however, the Nod fusion is not enriched at
the anterior margin in stage 11 (Fig.
2K), whereas the earlier ring-like localization pattern is
indistinguishable from the wild-type pattern (data not shown). In wild-type
stage 11 oocytes, microtubules extend from the center of the anterior cortex
towards the lateral margin, whereas these microtubules are strongly reduced in
Grip128 mutant oocytes (Fig.
2G,H). By contrast, the subcortical microtubule array, which has
been proposed to mediate cytoplasmic streaming
(Theurkauf and Hawley, 1992),
and cytoplasmic streaming itself appear to be normal in Grip128
mutant stage 11 oocytes (compare Movies 1 and 2 in the supplementary
material). Furthermore, nuclear migration and the organization of the
microtubule cytoskeleton in oocytes prior to stage 10b are normal in
Grip128 mutant oocytes (data not shown). We conclude that Grip128,
Grip75 and Tub37C establish a set of microtubules, which are presumably
nucleated from the anterior pole and are essential for bcd RNA
localization at stage 10b to 11.
To determine whether the defect in RNA localization is specific to bcd RNA, we analyzed the distribution of other localized transcripts such as osk, grk and orb RNAs. Both osk and grk RNA localization is unaffected in Grip128 mutants throughout oogenesis (see Fig. S1A-D in the supplementary material). Similarly, the localization of orb RNA is not disturbed in Grip128 mutant oocytes, as the RNA initially localizes in a bcd-like pattern and is then lost from the anterior cortex during stage 10 in wild-type and mutant oocytes (see Fig. S1E,F in the supplementary material). Taken together, the observed microtubule defect does not result in a general defect in RNA localization.
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TuRC during oogenesis-tubulin containing complexes
has not been analyzed in detail. More importantly, it is unclear whether a
TuRC can form in Grip75 or Grip128 mutants.
As a first step towards the characterization of ovarian TuRCs, we
analyzed the expression of the Grips 163, 128, 75, 91 and 84, as well as
Tub37C (Fig. 3A).
Additionally, we analyzed Swa expression as Swa and the TuRC have been
shown to interact (Schnorrer et al.,
2002). Both in wild-type and swaVA11 ovaries,
all TuRC components we tested were present, suggesting that early
embryonic TuRCs are similar in composition to ovarian TuRCs. In
Grip128326 and Grip75175 ovaries, the
TuSC components were present in equal amounts as in wild-type ovaries,
whereas the levels of Grip163 were reduced
(Fig. 3A). In
Grip75175 ovaries, Grip128 levels were lower, and less
Grip75 was present in Grip128326 ovaries compared with
wild type (Fig. 3A). A
reduction in the levels of Grip163 and Grip128 has also been observed in S2
cells depleted for Grip75 (Verollet et
al., 2006). All of the TuRC subunits were stable in
Tub37C139 mutants
(Fig. 3A), which is a null
allele (N.V., I.K. and C.N.-V., unpublished). These data suggest that the cap
subunits of the TuRC depend on each other for their stability in
ovaries.
Next, we immunoprecipitated Tub37C-containing complexes from ovarian
extracts using an antibody specific to Tub37C. From wild-type extract,
Tub37C co-immunoprecipitated with Grip91, Grip84, Grip163, Grip128 and
Grip75 (Fig. 3B). In addition,
Grip91 and Grip84 co-immunoprecipitated with Tub37C from
Grip75175 or Grip128326 ovarian
extracts, thus the TuSC forms normally in these mutants.
We wondered whether the TuSC might still provide
microtubule-nucleating activity in vivo or whether a ring complex assembles
with an incomplete cap structure. The latter possibility was already supported
by co-immunoprecipitation, which showed that some Grip128 was associated with
the TuSC components in the Grip75 mutant. As
immunoprecipitation experiments do not reveal the sizes of -tubulin
containing complexes, we performed sucrose density gradient centrifugation of
wild-type, Grip75175 and Grip128326
ovarian extracts (Fig.
3C-E).
Tub37C and Grip128 were present in high molecular weight fractions
in wild-type ovaries (Fig. 3C,
fractions 9-14), as has been described for embryonic Tub37C and Grip128
(Gunawardane et al., 2000;
Moritz et al., 1998;
Oegema et al., 1999). Grip128
and -tubulin are part of at least two differently sized complexes of
40 S and 60 S. The embryonic TuRC has been shown to have a
size of 37 S (Moritz et al.,
1998), thus the 40 S complex in ovarian extract could correspond
to the TuRC. The nature of the larger complex is unknown, but it is
possible that it consists of the TuRC in association with attached MTOC
material. Further experiments are necessary to resolve these issues. Both
complexes are sensitive to high salt concentrations (e.g. 500 mM KCl; data not
shown). Grip128 and -tubulin are also present as low molecular weight
entities, which presumably correspond to the TuSC and monomeric
Grip128. We also detected a 40 S complex in extracts of
Grip75175 or Grip128326 ovaries
(Fig. 3D,E, fractions 9-10),
whereas the larger complex is only present in very small quantities. High salt
concentrations lead to the disassembly of these complexes (data not shown). In
S2 cells depleted for cap components, severely reduced levels of TuRC
have been observed (Verollet et al.,
2006). We also see a reduction in the amount of the large
complexes, albeit less severe, which is presumably due to the less stringent
salt concentration we use for our experiments, as we noted that the mutant
complexes are more labile than the wild-type TuRC. Although we cannot
prove with certainty that the 40 S complexes in Grip75 and
Grip128 mutants are indeed incomplete TuRCs, this is a likely
possibility because they are similar in size to the wild-type TuRC and
because of the presence of the TuSC component -tubulin and the
TuRC component Grip128 in Grip75 mutant complexes. The mutant
complexes may still be capable of nucleating microtubules, providing an
explanation for the observed viability of Grip75 and Grip128
mutants.
Grip128 and Grip75 are required for meiosis in females
To better understand the function of Grip75 and Grip128,
we analyzed processes other than bcd RNA localization that depend on
these genes. In Grip75 or Grip128 mutant eggs, we did not
detect any nuclear divisions by DIC microscopy or DAPI staining (data not
shown), which could be due to either meiotic or mitotic defects. To determine
whether meiosis I was impaired in Grip75 and Grip128
mutants, we stained stage 14 oocytes with DAPI and analyzed the chromosome
arrangement. In oocytes, meiosis is arrested at metaphase I until the egg is
laid (King, 1970). In
wild-type, Grip75175 and Grip128326
oocytes, chromosomes were arranged in a variable but symmetric fashion
(Fig. 4A-C)
(Theurkauf and Hawley, 1992).
Thus, spindle formation in meiosis I appears normal in Grip75 and
Grip128 mutants.
|
). In Grip75175 and
Grip128326 eggs, the first meiotic spindle is anastral as
in wild type and appears to function properly
(Fig. 4D-F). By contrast,
meiosis II is severely disrupted. The central array of microtubules is absent,
and instead either two anastral spindles formed, which were not properly
aligned to each other and to the cortex, or the spindles were strongly
disorganized (Fig. 4H,J; data
not shown). In older oocytes, the chromosomes dispersed and were associated
with small, often misshaped, anastral spindles. Our results suggest that
either the central MTOC is not present or that it does not organize
microtubules in Grip75 and Grip128 mutant eggs.
Grip128 and Grip75 in male meiosis
Both Grip128 and Grip75 mutants are not only female
sterile but also male sterile. We analyzed spermatogenesis by phase contrast
microscopy in wild-type, Grip128, Grip75 and double mutant
spermatocytes (Fuller, 1993).
At the onion stage, Grip75175,
Grip128352 and the double mutant spermatids often
displayed a Nebenkern twice the size of a regular Nebenkern, which was
associated with two nuclei (Fig.
5B,C; see Table S1 in the supplementary material). Occasionally,
we observed some nuclei that were smaller than normal. The mitotic divisions
prior to meiosis were not severely affected in single or double mutant males,
as we did not observe pre-meiotic cysts with fewer than 16 cells (data not
shown).
These observations are further supported by ultrastructural analysis. In ultra-thin sections of wild-type testes, each flagellum contains the axoneme and the associated mitochondrial derivative (Fig. 5D). However, we often observed flagella with two axonemes in Grip75175 and Grip128352 spermatids (Fig. 5E,F). Our data suggest that Grip75 and Grip128 might not be essential for mitotic divisions in the male germline, but that both proteins are crucial in male meiosis. More specifically, cytokinesis is impaired in male meiosis and, occasionally, chromosome segregation defects occur. However, we have not vigorously excluded functions of Grip75 and Grip128 in pre-meiotic spermatocytes, therefore the observed meiotic phenotypes could also be due to unnoticed mitotic defects.
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Grip128 and Grip75 in sperm morphogenesis
Grip75 and Grip128 mutant males are completely sterile,
even though the cytokinesis defects in these mutants are not fully penetrant.
We therefore analyzed sperm morphogenesis to identify further functions of
Grip75 or Grip128.
|
). In
Grip128 and Grip75 mutant males, we did not observe sperm in
the seminal vesicle. Elongated sperm were present in mutant testes, but they
were not motile (data not shown). We analyzed chromosome and microtubule
distribution in Grip128352 and
Grip75175 spermatids. Whereas nuclei in wild-type
spermatids are packed at one end of the sperm bundle, the nuclei are dispersed
along the entire sperm bundle in Grip128352 and
Grip75175 testes (Fig.
6A-C). In wild-type spermatids, -tubulin is localized at
the junction between the nucleus and the elongating flagellum
(Fig. 6D)
(Wilson et al., 1997).
Strikingly, in Grip75175 and
Grip128352 spermatids, the association of -tubulin
with the nucleus was frequently lost (Fig.
6E,F), suggesting that the axoneme is not tightly attached to the
nucleus in these mutants.
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In conclusion, Grip128 and Grip75 mediate the attachment of -tubulin
to the nucleus, which is necessary for alignment of the nuclei at one end of
the sperm bundle. However, they are not necessary to build the complex axoneme
structures nor are they required for axoneme maturation.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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TuRC in microtubule nucleation has been studied
extensively by biochemical assays and electron microscopy. However, for many
of the TuRC components, an understanding of their function in the
context of an organism has not yet emerged. We show that components of the
TuRC, which were thought to be required for microtubule nucleation, can
have restricted and distinct functions. Our analysis of Grip128 and
Grip75 mutants suggests that the TuRC cap structure influences
the function of microtubules involved in bcd RNA localization during
oogenesis, meiosis in males and females, as well as sperm morphogenesis. In
Grip128 and Grip75 mutants, a TuRC seems to assemble
and to provide basic TuRC functions, which are sufficient for the
viability of adult flies and thus for all the essential processes in somatic
cells. Our data support the view that Grip128 and Grip75 anchor the
TuRC at specialized MTOCs, allowing microtubules that are required for
a few distinct processes to tightly associate with specific MTOCs.
Grip128 and Grip75 are not essential for viability
TuRC function in microtubule nucleation is crucial for viability, as
mutations in Grip91/l(1)dd4, Grip84 and Tub23C are
lethal (Barbosa et al., 2000;
Colombie et al., 2006;
Sunkel et al., 1995). 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 TuRC and that the TuRC 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
(Verollet et al., 2006), but
the cap components are not absolutely essential for mitotic progression. This
is not surprising as even mutants with centrosomal defects can survive
(Martinez-Campos et al.,
2004). Furthermore, -tubulin is recruited to centrosomes in
Grip75 or Grip128 mutant spermatocytes, Grip75
mutant neuroblasts and in S2 cells depleted for cap components (N.V. and
C.N.-V., unpublished) (Verollet et al.,
2006), showing that -tubulin targeting to the centrosome
does not depend on cap components. It has been proposed that -tubulin
can be recruited to centrosomes as part of the TuSC, as the amount of
large -tubulin-containing complexes is severely reduced in cells
depleted for cap components (Verollet et
al., 2006). Using buffers with lower salt concentrations, we
observe large -tubulin containing complexes in Grip75 and
Grip128 mutants, albeit in reduced amounts compared with wild type.
It is likely that these complexes are indeed TuRCs that lack parts of
the cap structure, as they are similar in size to the TuRC; in
addition, Grip128 is present in Grip75 mutant complexes. The mutant
TuRCs might still be capable of nucleating microtubules.
Whether -tubulin forms TuSCs or incomplete TuRCs, the
cap subunits are dispensable for microtubule nucleation and -tubulin
recruitment to centrosomes (this study)
(Verollet et al., 2006).
Moreover, a TuRC 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.
Overlapping functions of individual TuRC-specific subunits
In Drosophila, it is not known whether individual TuRC
complexes vary in their subunit composition and whether the
TuRC-specific subunits have similar functions. The human TuRC
has been shown to contain all of the described subunits
(Murphy et al., 2001). We and
others show that the cap components Grip75, Grip128 and Grip163 depend on each
other for their stability (Verollet et
al., 2006). Furthermore, individual depletion of Grip75, Grip128
or Grip163 results in a similar increase of the mitotic index in treated cells
(Verollet et al., 2006).
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.
By contrast, Grip71 appears to have a distinct function. On the one hand,
depletion by RNAi does not impair protein levels of the other
TuRC-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
(Verollet et al., 2006).
A microtubule-anchoring function of the TuRC cap structure
Genetic and cell biological data suggest that an intact cap structure is
not necessary for microtubule nucleation (this study)
(Verollet et al., 2006); thus,
the function of the cap is still in question. It could be required for
efficient assembly of the TuRC, 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.
Mutants with global defects in microtubule function such as hypomorphic
tub84B mutants show a wide range of phenotypes such as
polyphasic lethality, cuticle defects, short life span and sterility
(Matthews and Kaufman, 1987).
Similarly, hypomorphic Grip91/l(1)dd4 mutants are lethal and display
both mitotic and meiotic defects in spermatogenesis
(Barbosa et al., 2003;
Barbosa et al., 2000). As
Grip75 and Grip128 mutants show very specific phenotypes, a
function for the TuRC 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.
The Drosophila pericentrin-like protein D-PLP recruits or anchors
-tubulin to centrosomes, possibly by direct interaction with
TuSC components (Kawaguchi and
Zheng, 2004; Martinez-Campos
et al., 2004). Interestingly, D-PLP is only required for efficient
anchoring of -tubulin to the centrosome in early phases of mitosis,
suggesting that a D-PLP independent pathway can recruit and anchor centrosomal
components (Martinez-Campos et al.,
2004). Maybe D-PLP and the TuRC cap structure act
redundantly in anchoring -tubulin at the pericentriolar material during
mitosis.
Additionally, microtubule motors focus microtubules at the mitotic
centrosome. Inhibition of the dynein-dynactin complex results in disorganized
spindles that lack well-focused poles
(Gaglio et al., 1997), while
analysis of Dhc64C mutations in Drosophila suggests that
dynein is required for the attachment of spindle poles at centrosomes
(Robinson et al., 1999). The
kinesin-related Ncd is a minus-end directed microtubule motor that also
functions in spindle assembly during mitosis
(Endow et al., 1994). Depletion
of Ncd by RNAi in S2 cells results in frequent release of microtubules from
the spindle pole (Goshima and Vale,
2003).
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 (Martinez-Campos et al.,
2004); however, a possible function in -tubulin anchoring
in male meiosis II is difficult to address because of the centriolar defects.
Ncd organizes the female meiosis I spindle
(Matthies et al., 1996) and
also localizes to the meiosis II spindle. ncd mutants do not form a
structured central aster in meiosis II
(Endow and Komma, 1998), but
this could be a consequence of defects in meiosis I. We propose 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 TuRC cap subunits Grip128 and Grip75. Hence, these
proteins allow the organization of distinct microtubule populations at
particular positions in complex cells, independently of centrosomes.
Grip128 and Grip75 at specialized MTOCs
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
(Verollet et al., 2006). 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.
Grip128, Grip75 and Tub37C participate in the formation of a new
MTOC at stage 10b, which directs the relocalization of bcd RNA during
stage 10b (this study) (Schnorrer et al.,
2002). They are specifically involved in bcd RNA
localization, as 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 (Schnorrer et al.,
2002). 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
(Pokrywka and Stephenson,
1995); however, orb RNA is correctly localized in
Grip128 mutant oocytes. These data argue against a general
microtubule impairment in Grip128 mutants.
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 (Matthies et al.,
1996). 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
(Riparbelli and Callaini,
2005). It contains -tubulin, whereas the distal poles are
devoid of -tubulin (Endow and Komma,
1998; Matthies et al.,
1996). 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. We
favor the latter hypothesis, as 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
(Riparbelli and Callaini,
2005; Riparbelli et al.,
2000).
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
(Gonzalez et al., 1998).
Centrioles in spermatocytes are large and associated with very little
pericentriolar material when compared with mitotic centrioles
(Fuller, 1993;
Riparbelli et al., 2002).
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.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/20/3963/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Institute of Molecular Pathology, Dr Bohr-Gasse 7, 1030
Wien, Austria 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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