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Maelstrom coordinates microtubule organization during Drosophila
oogenesis through interaction with components of the MTOC
Kaoru Sato, Kazumichi M. Nishida, Aoi Shibuya, et al.
Genes Dev. 2011 25: 2361-2373
Access the most recent version at doi:10.1101/gad.174110.111

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Maelstrom coordinates microtubuleorganization during Drosophila oogenesis
through interaction with componentsof the MTOC
Kaoru Sato,1 Kazumichi M. Nishida,1 Aoi Shibuya,1 Mikiko C. Siomi,1,2 and Haruhiko Siomi1,3
1
Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan; 2Core Research for Evolutional
Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, JapanThe establishment of body axes in multicellular organisms requires accurate control of microtubule polarization.
Mutations in Drosophila PIWI-interacting RNA (piRNA) pathway genes often disrupt the axes of the oocyte. This
results from the activation of the DNA damage checkpoint factor Checkpoint kinase 2 (Chk2) due to transposon
derepression. A piRNA pathway gene, maelstrom (mael), is critical for the establishment of oocyte polarity in thedeveloping egg chamber during Drosophila oogenesis. We show that Mael forms complexes with microtubuleorganizing center (MTOC) components, including Centrosomin, Mini spindles, and gTubulin. We also show that
Mael colocalizes with aTubulin and gTubulin to centrosomes in dividing cyst cells and follicle cells. MTOC
components mislocalize in mael mutant germarium and egg chambers, leading to centrosome migration defects.
During oogenesis, the loss of mael affects oocyte determination and induces egg chamber fusion. Finally, we showthat the axis specification defects in mael mutants are not suppressed by a mutation in mnk, which encodes
a Chk2 homolog. These findings suggest a model in which Mael serves as a platform that nucleates other MTOC
components to form a functional MTOC in early oocyte development, which is independent of Chk2 activation
and DNA damage signaling.
[Keywords: maelstrom; microtubule; centrosome; piRNA; axis specification; Drosophila oogenesis]
Supplemental material is available for this article.
Received July 18, 2011; revised version accepted October 6, 2011.

The cellular processes that elaborate asymmetries along
the main body axes are essential to the development ofmulticellular organisms. In Drosophila, the body axes are
established during oogenesis through the differential
localization of a large number of specific transcripts, including bicoid (bcd), oskar (osk), and gurken (grk) mRNAs.
This localization depends on a polarized microtubule(MT) cytoskeleton and correlates with the formation of
an MT-organizing center (MTOC), a small organelle fromwhich most MTs grow (Ephrussi et al. 1991; Chasan andAnderson 1993; Neuman-Silberberg and Schupbach 1993;
Theurkauf et al. 1993; Pokrywka and Stephenson 1995;
van Eeden and St Johnston 1999; Riechmann and Ephrussi
2001; Januschke et al. 2006; Steinhauer and Kalderon
2006). Thus, the formation of the axis depends on upstream
cues that lead to the polarization of MTs.
The Drosophila ovary consists of 16–20 independent
strings of egg chambers called ovarioles, which are the3
Corresponding author.
E-mail awa403@z2.keio.jp.
Article is online at http://www.genesdev.org/cgi/doi/ 10.1101/gad.174110.111.

functional units of oogenesis. Oogenesis starts with anasymmetric division of a germline stem cell, which occurs
at the anterior tip of the germarium located at the anterior
tip of an ovariole. This gives rise to a new stem cell anda differentiating daughter cell, the cystoblast (Supplemental Fig. S1). Cystoblasts in turn undergo four mitoses
to generate a cluster of 16 germ cells, which are interconnected by cytoplasmic bridges, called ring canals (Spradling1993). This cluster, named the cyst, is surrounded by amonolayer of somatic follicle cells to form an egg chamber,which then exits the posterior end of the germarium andproceeds through the 14 stages of oogenesis as it moves
posteriorly toward the oviduct (Spradling 1993). Two
germ cells have four ring canals, and one of these cells
is selected to differentiate into the oocyte. This cellremains arrested in meiotic prophase I and moves to theposterior of the cyst in region 3 of the germarium,
whereas the other germ cells undergo endoreplication to
generate polyploid nurse cells (Spradling 1993). Whichcell adopts the oocyte fate may depend on the asymmetric
distribution of the fusome, a continuous vesicular organelle linking the 16 cells via the ring canals (Lin et al. 1994;

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Sato et al.

Lin and Spradling 1995). The fusome is partitioned asymmetrically during cystoblast divisions, which requires its
stable association with MTs (Grieder et al. 2000). It has
been hypothesized that the cell that inherits the mostfusome material is the one that will become the oocyte.
Shortly after oocyte specification, nurse cell centrosomes
migrate toward the oocyte, where they eventually accumulate at the posterior of the nucleus, thus defining the
major MTOC of the entire cyst (Mahowald and Strassheim
1970; Grieder et al. 2000). As a cyst moves in region 2b of
the germarium, the fusome is replaced by a polarized MT
network emanating from a single MTOC that resides at
the anterior of the future oocyte. This network extends
through the ring canals into the nurse cells (Grieder et al.
2000) and is polarized such that the major routes oftransport are directed from the nurse cells toward theoocyte (Theurkauf et al. 1992; Clark et al. 1994, 1997). Thisasymmetric arrangement of the germ cells generates thefirst anterior–posterior (A–P) polarity during development.
Thus, initial polarization of the oocyte during the oocyte
specification phase requires MTs and correlates withthe formation of the MTOC in the anterior of the cell
(Theurkauf et al. 1993).
In region 3, when oocyte positioning is completed,a reorganization of the oocyte MT network takes place,which shifts the anterior MTOC to the posterior pole
(Clark et al. 1997). This polarized MT network is required
for asymmetric localization of Grk, a TGF-b homolog(Januschke et al. 2006). The A–P axis of the embryo isthen polarized by two signaling events. At stage 6, theGrk signal from the oocyte induces the adjacent follicle
cells to adopt a posterior rather than an anterior fate. At
stages 7–8, the oocyte MT cytoskeleton undergoes a dramatic reorganization in response to an unknown signal
from the overlying posterior follicle cells (Theurkauf
et al. 1992; Januschke et al. 2006). The posterior MTOC
is disassembled and, accompanying this process, the oocyte
nucleus moves away from the posterior pole and localizes
at the anterodorsal corner of the oocyte (Januschke et al.
2006). MTs then become repolarized, which directs thetransport of mRNAs encoding determinants importantfor embryonic development; for example, bcd mRNA istransported to the anterior of the oocyte and osk mRNAis transported to the posterior of the oocyte (Brendza et al.
2000; Schnorrer et al. 2000; Arn et al. 2003). At the sametime, grk mRNA localizes to the anterodorsal cortex of
the oocyte, leading to Grk signaling to the dorsal follicle
cells (Neuman-Silberberg and Schupbach 1993, 1996).
Thus, the oocyte and the surrounding eggshell are patterned by the reciprocal germline-to-soma signaling cascade. Although the process of early oocyte polarization
clearly depends on an intact MT network and correlateswith the formation, shift, and disassembly of the MTOC,
the mechanisms involved are not well understood.
PIWI-interacting RNAs (piRNAs), a class of small
RNAs associated with PIWI proteins of the Argonautefamily, silence transposable elements in animal germ
cells (Ghildiyal and Zamore 2009; Malone and Hannon
2009; Siomi et al. 2011). In Drosophila ovaries, piRNAs
associate with the PIWI subfamily of Argonaute proteins

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GENES & DEVELOPMENT(AGO3, Aubergine [Aub] and Piwi) to form the effector
piRISC, which represses retrotransposons and preserves
the integrity of the genome (Aravin et al. 2001; Kalmykova
et al. 2005; Saito et al. 2006; Vagin et al. 2006; Brennecke
et al. 2007; Gunawardane et al. 2007; Nishida et al. 2007;
Li et al. 2009; Malone et al. 2009). A large number of genes
have been shown to be involved in piRNA production in
Drosophila ovaries. The majority of these piRNA pathway
genes are required for assembly of nuage, a perinuclear
structure found at the cytoplasmic face of the nuclear
envelope in animal germline cells, where multiple components of the piRNA pathway are enriched, enabling
piRNA processing to occur (Lim and Kai 2007; Li et al.
2009; Malone et al. 2009). Mutations in these piRNA
pathway genes often disrupt axis formation and give rise
to disorganized MTs in ovaries, which ultimately lead tothe production of spindle-shaped eggs (Gillespie and Berg
1995; Wilson et al. 1996; Clegg et al. 1997; Lasko andAshburner 1988; Styhler et al. 1998; Tomancak et al.
1998; Cook et al. 2004; Klattenhoff et al. 2007; Pane et al.
2007; Klattenhoff and Theurkauf 2008). Indeed, many
piRNA pathway genes, including aub, armitage (armi),
maelsrom (mael), spindle-E (spnE), vasa (vas), and zucchini(zuc), had been put in the spindle class of genes before
their involvement in piRNA production was revealed(Gillespie and Berg 1995; Wilson et al. 1996; Clegg et al.
1997; Lasko and Ashburner 1988; Styhler et al. 1998;
Tomancak et al. 1998; Cook et al. 2004; Pane et al. 2007).
Mutations in spindle class genes are highly pleiotropic,
resulting in axis patterning defects, which is attributed to
failures in MT polarization and mRNA localization (Tearleand Nüsslein-Volhard 1987; Schupbach and Wieschaus
1991; Gonzalez-Reyes et al. 1997). armi, aub, spn-E, andvas mutant oocytes display defects in MT organization,
including assembly of the MTOC (Styhler et al. 1998;
Cook et al. 2004; Klattenhoff et al. 2007). These findings
indicate that in piRNA pathway mutants, the MT network
is not polarized, which disrupts the Grk signaling and theinitiation of oocyte patterning. Thus, it is possible thatpiRNA pathway genes are involved in the machinery that
impacts MT organization, thereby indicating a link between piRNA production and the polarization of MTs.
In the majority of piRNA pathway mutations, theearliest phenotype is a persistent DNA damage in germline cells of the germarium, probably due to transposon
derepression and mobilization, which can lead to DNA
breaks (Klattenhoff and Theurkauf 2008). The axis-specificdefects associated with these mutations are suppressed bymutations in mei-41 and mnk, which encode ATR andCheckpoint kinase 2 (Chk2) kinases that function in DNA
damage signaling (Klattenhoff et al. 2007). Significantly,
the same mutations in mei-41 and mnk also restore
MTOC formation during early oogenesis (Klattenhoff
et al. 2007). How checkpoint activation causes changes
in the cytoskeletal network is currently unknown; however, these findings suggest that the axis specification
defects associated with piRNA pathway mutations area secondary consequence of DNA damage signaling.
The mael gene was originally identified based on its
role in Drosophila oocyte axis specification (Clegg et al.

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Mael coordinates MT organization

1997). mael mutants exhibit mRNA localization patterns
similar to grk mutants in stages 8–10, accumulating bcd
mRNA at both poles and osk mRNA in the center of theoocyte (Clegg et al. 1997). Mutations in mael also prevent
efficient grk translation in stages 2–6 and disrupt bothoocyte nucleus migration to the anterodorsal corner andposterior localization of the MTOC in stage 2–6 oocytes(Clegg et al. 2001). Thus, failure of early Grk signaling in
the oocyte has been proposed to be the underlying causeof A–P defects in mael mutants. Although Mael contains
a domain with weak similarity to a high-mobility group(HMG) box and a domain called the Mael domain withweak homology with the DnaQ-H 39-to-59 exonucleasefamily (Zhang et al. 2008), its biochemical functions inboth axis specification and piRNA production remains to
be clarified. Mael localizes to the nucleus as well as to thenuage (Findley et al. 2003; Lim and Kai 2007). The nuage
localization of Mael depends on other piRNA genes,
including vas, aub, and spn-E (Findley et al. 2003; Lim
and Kai 2007).
In the course of biochemically characterizing the role ofMael in Drosophila ovaries, we found that Mael physicallyinteracts with MTOC components. These MTOC components mislocalize in the germarium of mael mutants,
leading to centrosome migration defects that are indicative of defects in MT organization. We show that the lossof mael affects oocyte determination and causes egg
chamber fusion. The patterning defects associated withmael mutants are not suppressed by mutation of mnk. We
propose that, through interaction with MTOC components, Mael contributes to the coordination of MT organization in early oogenesis, which is independent of Chk2
activation and DNA damage signaling and is, therefore,
probably independent of its function in piRNA production.

proteins were resolved by SDS–polyacrylamide gelelectrophoresis and visualized by silver staining (Fig. 1A).
The eluates were further analyzed by mass spectrometry
to identify protein partners that engage with Mael inthe ovary. Six proteins were identified as major Maelinteracting proteins: Centrosomin (Cnn), Drosophilatransforming acidic coiled-coil protein (D-TACC), Mini
spindles (Msps), Short spindle 2 (Ssp2), dNAT1 (CG3845),
and No circadian temperature entrainment (Nocte). In
animal cells, many of these proteins are associated withcentrosomes that act as MTOCs to nucleate MTs withuniform polarity (Kwon and Scholey 2004). Centrosomes
are composed of two centrioles that recruit a large numberof proteins collectively referred to as pericentriolar material (PCM). Cnn is a core centrosome protein required fornormal PCM organization and astral MT assembly(Megraw et al. 2001; Terada et al. 2003). Msps, a memberof the conserved MT-associated proteins, localizes to centrosomes and mitotic spindles and is required for the
integrity of mitotic spindles (Cullen et al. 1999; Cullen
and Ohkura 2001). D-TACC is also a centrosomal proteinthat interacts with MTs and is found at the spindle poles.
Complex formation of D-TACC with Msps has been
postulated to stabilize centrosomal MTs (Gergely et al.

Results
Mael forms complexes with MTOC components
To biochemically characterize its function, we produced
a specific monoclonal antibody against Mael. Western
blot analysis showed a discrete band in both ovary andcultured Schneider2 (S2) cells but not in mael-depleted S2cells (Supplemental Fig. S2A), demonstrating the specificity of the antibody. These results also indicated that
Mael expression is not germline-specific. Immunostaining
revealed that Mael in S2 metaphase cells is cytoplasmic
and concentrated at mitotic spindles (Supplemental Fig.
S2B,C). Lack of staining in mael-depleted S2 cells andmael mutant ovaries further indicated no off-target bindingof the antibody (Supplemental Fig. S2B,D). We performed
immunoprecipitation from ovary lysates with the antibody
in a buffer containing a strong detergent, Empigen, which
will disrupt most protein–protein interactions in vivo,
although antigen–antibody associations are maintained(Piñol-Roma et al. 1990). Mael was the only protein precipitated with the antibody under these conditions, further
confirming the specificity of the antibody (Supplemental
Fig. S2E). In a milder Digitonin-based buffer, Mael was
copurified with other protein factors. Mael-associatedFigure 1. Mael forms complexes with MTOC components inovaries. (A) Immunoprecipitation was performed from ovary
lysates using an anti-Mael monoclonal antibody. Proteins immunoprecipitated were stained with silver. Mass spectrometry analyses revealed that Nocte, Msps, D-TACC, dNAT1, Cnn, and Ssp2
coimmunoprecipitated with Mael. ‘‘n.i.’’ indicates nonimmune
IgG used as a negative control. (B) Western blotting on the Mael
immunoprecipitated complex in A using anti-Msps, anti-D-TACC,
and anti-Cnn confirmed Mael binding with Msps, D-TACC,
and Cnn. Nonimmune IgG (n.i.) was used as a negative control.
(C) Immunoprecipitation was performed from wild-type and cnn
mutant ovary lysates using an anti-Mael monoclonal antibody.
cnnHK21 is a cnn-null allele with a nonsense mutation thattruncates the protein at amino acid 106 (Megraw et al. 1999).
Mael binds with gTub and Msps in a Cnn-independent manner.

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Sato et al.

2000; Lee et al. 2001). Cnn and the Msps–D-TACC
complex are required for efficient MTOC formation.
Knockdown of ssp2 produces a short spindle phenotype,suggesting that this gene is also required for the integrityof mitotic spindles (Goshima et al. 2007). The nocte geneencodes a large glutamine-rich protein with unknown
function and is involved in maintaining the structure ofcilia in circadian clock neurons (Sehadova et al. 2009).
dNAT1 is a fly homolog of the vertebrate translational
regulator NAT1/DAP5/p97, an eIF4G family member(Yoshikane et al. 2007).
The finding that Mael forms a complex in vivo withcomponents of the MTOC suggests that Mael is also
a component of the MTOC. Western blot analyses on the
immunoprecipitates confirmed that Cnn, Msps, and
D-TACC exist in the Mael complex (Fig. 1B; Supplemental
Fig. S2F). The immunoprecipitates from mael mutant
ovaries showed no bands corresponding to Cnn, Msps, or
D-TACC (Supplemental Fig. S2F). These data show that
Mael specifically forms complexes with components of
the MTOC and suggest its involvement in normal MTOC
formation as an MTOC component. To further confirm
our findings, we examined whether Mael interacts withother MTOC proteins in ovaries. gTubulin (gTub) is
a conserved component of all MTOCs and is required
for them to nucleate MT polymerization from heterodimers of aTub and bTub (Wiese and Zheng 1999). We
performed Western blot analysis using an anti-gTub
antibody on immunopurified Mael complexes and foundthat Mael indeed interacts with gTub (Fig. 1C). AlthoughgTub was no longer recruited to the centrosome in theabsence of Cnn in Drosophila S2 cells (Goshima et al.
2007), gTub associated with Mael in cnn mutant ovaries,
indicating that the association between gTub and Mael inthe ovary is Cnn-independent (Fig. 1C). This also suggeststhat gTub–Mael complexes are formed before they become
stable components of the MTOC. These results are also inaccordance with previous genetic analysis showing thatmael is required for proper positioning of the MTOC
during oogenesis (Clegg et al. 1997, 2001).
Mael colocalizes with aTub and gTubin the germarium
Prior studies showed that in the germarium, Mael waspresent in all regions with diffuse nuclear and cytoplasmic
localization, and its accumulation in the nuage became
evident in region 3 (Findley et al. 2003). As a starting point
for defining the role of Mael in the MTOC, we re-examined
its localization using the antibody we produced andreasoned that Mael could colocalize with components of
the MTOC in the ovary. Mael was highly concentrated individing cells in the germarium, as judged by the coexpression of the mitotic marker, phospho-histone H3 (Ser10) (pH3) (Fig. 2; Adams et al. 2001). Lack of staining inmael mutant ovaries indicated that the signals observed
depend on the presence of Mael (Supplemental Fig. S2D).
In region 1 of the germarium, Mael accumulated in discrete regions of dividing cyst cells and colocalized withaTub, indicating that Mael was concentrated at the MT

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Figure 2. Mael colocalizes with aTub in the germarium. (A–A09)
Ovaries were immunostained with anti-Mael, anti-aTub, andanti-pH3 antibodies. pH3 is a mitotic marker. In region 1 of thegermarium, Mael accumulates in discrete regions of dividing cyst
cells. The Mael signals overlaps with the gTub signals, suggestingthat Mael is localized to the MT spindles in early cyst cells. In
region 2, Mael is localized to granules, in which gTub wasundetected. In region 3, Mael is accumulated in the nuage, whichis also devoid of gTub. A Mael signal in a somatic precursor cellis shown by an arrow. (B–B09) Magnified views of dividing cyst
cells in A–A909. (C–C09) Magnified views of the early egg chamber
in A–A09.

spindles of early cyst cells (Fig. 2A,B). In region 2, Maelbecame localized to granules, which did not colocalize
with aTub (Fig. 2A). In region 3, Mael became localized to
the nuage, which did not colocalize with aTub (Fig.
2A,C). Thus, Mael colocalized with aTub to centrosomesin early dividing cyst cells and then later became accumulated at the nuage in the germarium. Although Maelis known to colocalize with components of the piRNA
pathway in the nuage, mass spectrometry analyses of thecomplex that we isolated revealed no known proteinsfunctioning in the piRNA pathway, at least as major
components (Fig. 1A). We also failed to detect PIWI
proteins in the complex by Western blot analysis (Supplemental Fig. S3A). Reciprocal immunoprecipitation experiments with antibodies against PIWI proteins further
showed that PIWI proteins do not interact with Mael orother MTOC components (Supplemental Fig. S3B–D).
Thus, these results suggest that although Mael changes
its subcellular localization during early oogenesis, themajority of the protein in ovaries physically interacts
with MTOC components (Fig. 1A) but not with components of the piRNA pathway.
To confirm its centrosomal localization in region 1 ofthe germarium, we performed double labeling of Maeland gTub in the germarium. gTub is known to be present
on the centrosomes in the germarium and on migrating
centrioles (Mahowald and Strassheim 1970; Bolı́var et al.

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Mael coordinates MT organization

2001). Double labeling of Mael and gTub showed theiroverlap in cyst cells in region 1 (Fig. 3A). Therefore, Maelwas concentrated at centrosomes of dividing cyst cells
and colocalized with gTub and aTub in region 1 of thegermarium. Together, these findings establish that Maelis a component of centrosomes in dividing cyst cells.
Mael is concentrated in the centrosomal region
during follicle cell divisions
Mael was highly expressed in somatic precursor cells (Fig.
2A, arrow; Supplemental Fig. S4). Later, Mael did notaccumulate in nuage-like structures in follicle cells but
was concentrated at MT spindles of dividing follicle cells
in egg chambers, as judged by colocalization with aTub
(Fig. 3B). Therefore, Mael colocalizes with these proteinsin both dividing cyst cells and dividing follicle cells in theovary. These findings suggest that Mael is involved incentrosomal function during Drosophila oogenesis.
Protein components of the MTOC are mislocalized
in mael egg chambers
An early event in oocyte differentiation is the establishment of a single MTOC in the cyst. At the end of fourcystocyte divisions, the centrosomes are inactivated andthe centrioles are transported into the pro-oocyte wherea single MTOC in the cyst is established. From this focus
in the oocyte, MTs emanate into all of the 15 nurse cells.
This polarized MT array is essential for the transport ofmaternal factors required for oocyte differentiation anddevelopment (for reviews, see Cooley and Theurkauf 1994;
Knowles and Cooley 1994; Mahajan-Miklos and Cooley
1994; Theurkauf 1994). Centrosomes have been reported
to migrate into the pro-oocyte during cyst developmentin the germarium. Our finding that Mael interacts withcentrosome components suggests that centrosome migration may be perturbed in mael mutant egg chambers.
To test this, we examined the localization of gTub and
Cnn in the mael germarium (Fig. 4). In the wild-typegermarium, gTub accumulated at dot-like structures,
which were presumably centrosomes in region 1. These
started to migrate into pro-oocytes in region 2 and thenaccumulated in pro-oocytes in region 3. gTub also
accumulated at dot-like structures in region 1 of themael germarium, but it remained at these structures anddid not accumulate in pro-oocytes in regions 2–3 (Fig.
4A). In the wild-type germarium, Cnn accumulated in
the cytoplasm of germline stem cells in region 1, but its
expression in regions 2–3 was barely detected. Cnn was
also accumulated in the cytoplasm of germline stem
cells in region 1 of the mael germarium, but its expression
was detected at significant levels in pro-oocytes in regions
2–3 (Fig. 4B). Thus, both gTub and Cnn were mislocalized
and ectopically expressed in the mael germarium.
We also observed mislocalization of gTub and Cnn inoocytes in developing mael egg chambers. In the wild
type, gTub accumulated in oocytes with a punctuatedpattern (Fig. 4C,E). However, gTub was highly concentrated at the posterior region of mael oocytes (Fig. 4C,E).
Cnn localized to the posterior region of oocytes in stages
6–8 of mael egg chambers as in the wild type (Fig. 4D,E).
However, Cnn was detected with granules in mael egg
chambers. In addition, gTub and Cnn were ectopically
expressed in ;39% and 29%, respectively, of all mael egg
chambers. Some egg chambers contained two strong
signals at both poles, while others contained misplaced
oocytes (Fig. 4C–E). We also occasionally observed mael
egg chambers that contained abnormal accumulationof gTub in the nurse cells (Fig. 4C). These observations
provide a functional involvement of mael in the proper
localization and expression of MTOC proteins during
Drosophila oogenesis.
Loss of mael function affects the distribution
and dynamics of oocyte MTs

Figure 3. Mael is localized to mitotic spindles in the dividing
cyst and follicle cells. (A–A09) Immunofluorescence was performed using anti-Mael, anti-aTub, and anti-gTub antibodies.
Mael is colocalized with aTub and gTub at mitotic spindles ofdividing cyst cells. (B–B09) Immunofluorescence was performed
using anti-Mael, anti-aTub, and anti-pH3 antibodies. In dividing
follicle cells (pH3-positive cells; indicated by arrows), Mael iscolocalized with aTub to mitotic spindles. Bars, 20 mm.

The dynamics of MT accumulation and the temporal–
spatial changes in the MTOC have been observed in thedeveloping oocyte by staining MTs with antibodies
against aTub (Theurkauf et al. 1992). From stage 1 tostage 6, MTs extend into the nurse cells from the MTOC,
situated at the posterior of the oocyte (Theurkauf et al.
1992, 1993). Mislocalization and ectopic expression ofgTub and Cnn in mael egg chambers prompted us to
examine the MT cytoskeleton in mael ovaries. We compared the MT cytoskeleton of the germariums and of stage

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Figure 4. Protein components of the MTOC
are mislocalized in mael mutant ovaries.
(A) gTub localization in the germarial region of wild-type (left panels) and maelmutant (right panels) ovaries. Anti-Orb antibody was used as a marker for developing
oocytes. gTub is accumulated in the oocyte
in the wild-type germarium (arrow), but
was absent from the mael mutant oocyte
in the germarium. (B) Cnn localization in
the germarial region of wild-type (left panels)
and mael mutant ovaries (right panels). Cnn,
which is undetectable in the oocyte in thewild-type germarium, was detected in theoocytes in the mael mutant germarium(arrows). (C) gTub localization in wild-type
and mael mutant egg chambers. In the wild
type, gTub is specifically accumulated at theanterior region in the oocyte. However, inthe mael mutant, gTub was often mislocalized to the posterior region of the oocytes. gTub was also ectopically accumulated
within the egg chambers (arrows) of mael.
(D) Cnn localization in wild-type and maelmutant egg chambers. Cnn, which is accumulated at the posterior region in the wild-type oocyte, was detected as speckles in themael mutant oocytes. Cnn was ectopically accumulated within the egg chambers (arrows). (E) The percentage of mael egg chambersthat show ectopic localization of Cnn and gTub. Bars, 20 mm.

1–8 egg chambers in mael and wild-type ovaries to look forphenotypic differences. In the wild-type, an MTOC was
detected at the posterior region of the stage 1 oocyte(region 3 in the germarium), whereas an MTOC was notdetected in the stage 1 mael mutant oocytes (Fig. 5A,B).
The MT cytoskeleton in the mael germarium was clearly
disorganized (Fig. 5B). In the wild type, aTub was concentrated in stage 3–6 oocytes, in the location where MTOCs
are formed (Theurkauf et al. 1992). MTs extended from an
MTOC at the posterior region of stage 3–6 oocytes throughthe ring canals and into nurse cells (Fig. 5C). However,high concentrations of MTs at the posterior region of stage
3–6 oocytes in mael mutant ovaries were not observed(Fig. 5C). This is not due to reduced expression of aTub inmael mutant ovaries, as judged by Western blot analysis(Supplemental Fig. S5).

At stage 7, a shift in the polarity of MT accumulation
occurs in oocytes. At this time, the oocyte nucleus
migrates to the anterior of the oocyte. Nuclear migration
is an MT-dependent process that in many systems is dependent on MTOCs associated with the nucleus (Reinschand Gonczy 1998). After migration of the nucleus to a
position that will mark the anterior–dorsal position of thedeveloping oocyte, MTs are rolled up in the shape of a‘‘diaphragm’’ in the center of the oocyte. In stages 8–9, the‘‘diaphragm’’ has opened and MT bundles project from theanterior of the oocyte in the A–P direction in a horseshoelike pattern (Supplemental Fig. S1; Januschke et al. 2006).
In the wild type, in stages 7 and 8, an MTOC was normallyreorganized and the oocyte nucleus migrated anteriorly(Fig. 5C). However, in mael mutant oocytes, MT accumulation and reorganization were not observed (Fig. 5C).

Figure 5. MTOC formation is defective in maelmutant ovaries. MT distribution in wild-type andmael mutant (maelM391/Df ) oocytes was visualizedwith anti-aTub antibody (white). DNA was visualizedwith DAPI (blue). (A) MTs in the control germariumand stage 1 egg chamber. The MTOC was observed at
the posterior region of the oocyte in region 3 of thegermarium. A red asterisk indicates the nucleus of
the oocyte that is denoted by a dotted line. (B) MTs in
the mael mutant germarium and stage 1 egg chamber.
The MTOC was not detected when mael function
was lost. (C) MTs in stage 3–8 egg chambers in control
and mael mutant ovaries. The MTOC was detected at
the posterior region of stage 3–6 control oocytes, but
not in the mael mutant oocytes. In the wild type,MTs are then rolled up (‘‘diaphragm’’ pattern) during stage 7 (Supplemental Fig. S1) and project from the oocyte nucleus in an anterior–posterior direction at stage 8 (‘‘open diaphragm’’ pattern). This reorganization of MTs was not observed in the mael mutant. Bars, 20 mm.

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Mael coordinates MT organization

Thus, mael MT cytoskeletons are not organized to form
an MTOC even in the germarium and are unable toreorganize and rearrange into an elaborate pattern of
subcortical MT bundles that mediate oocyte nucleus
migration in stages 6–8 of the oocyte.
To gain further insights into the involvement of Mael inMT organization and dynamics, we examined cell divisionof Mael-depleted S2 cells. We observed that Mael depletion
delayed the progression to anaphase and thereby increased
the percentage of metaphase cells (;4% of the population)(Supplemental Fig. S6). These results, together with theconcentration of Mael at mitotic spindles in S2 cells(Supplemental Fig. S2D), suggest that Mael is required forthe efficient assembly of metaphase spindles in S2 cells
and further implicate Mael in MT organization.
Loss of mael function affects the number of germ cells
per egg chamber
Mael mutant ovaries have a defective MT cytoskeleton
with no clear MT accumulation or MTOC throughout
the early stages of the oocyte. In addition, a significant
number of mael egg chambers show ectopic expressionof gTub and Cnn. Thus, we re-examined the number andposition of the oocytes in mael mutant egg chambers.
We observed that ;27% of egg chambers in mael mutant
ovaries have misplaced oocytes (stained with anti-Orb

antibody) and ;5.5% of mael egg chambers have twooocyte-like nuclei with >15 nurse cells (Fig. 6A,B). In theegg chambers with supernumerary germ cells, an MTOC
was not detected in either of the two oocyte-like cells(Supplemental Fig. S7). The posterior localization of theoocyte is thought to be directed by cell sorting via DEcadherin (DE-cad) (Godt and Tepass 1998; González-Reyes
and St Johnston 1998). In mael mutant ovary egg chambers,
DE-cad did not appear to be enriched on the oocyte cortex
or apically concentrated in follicular epithelia comparedwith the wild type (Supplemental Fig. S8), indicating that
this abnormal localization of DE-cad may be responsible
for the misplaced oocytes in mael egg chambers.
We observed two types of egg chamber in mael mutants
with abnormal numbers of germ cells. Egg chambers with
>16 germ cells could be generated by either the fusion of
two egg chambers or mitotic abnormality of the cystoblast. A normal developing egg chamber contains 16 germ
cells with an oocyte and 15 ring canals in the egg chamber(Fig. 6C). If an egg chamber was fused with the anterior
egg chamber due to defective follicle cell development,
the fused egg chamber should have 32-cell cysts with 30
ring canals, which is twice the number of the wild type. It
is also possible that an egg chamber with 32-cell cysts
arises from an extra mitotic division, as reported for thetribbles gene mutation (Mata et al. 2000). In this case,however, the egg chamber should have 31 ring canals

Figure 6. Defective egg chamber development in the mael mutant. (A) Wild-type (top) and mael mutant (bottom) oocytes werevisualized by immunohistochemical analyses using an anti-Orb antibody (green). DNA was stained with DAPI (blue). In the wild type,the oocyte is localized at the posterior region of the egg chamber. In contrast, in the mael mutant, egg chambers possessing
a mislocalized oocyte (arrowheads) or two oocytes (arrows) were observed. (B) The percentage of abnormal egg chambers in the maelmutant. (C) The number of nurse cells and ring canals in the mael mutant egg chambers. Ring canals (red) and oocytes (green) werevisualized using PY20 and anti-Orb antibody, respectively. DNA was stained with DAPI (blue). (D) The population of abnormal egg
chambers in the mael mutants. NC, RC, and Oo indicate the number of nurse cells, ring canals, and oocytes, respectively. (Groups a–c)
See the text. Bars, 20 mm.

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(Supplemental Fig. S9; Huynh 2006). To investigate apossible mechanism in mael mutant ovaries that leads tothe production of egg chambers with >16 germ cells, we
examined the number of ring canals in mael egg chambers. About 5.6% of egg chambers showed 30 nurse cellswith 30 ring canals and two oocyte-like nuclei (30 + 2)
(Fig. 6C [panel b], D [panel c]). Therefore, these maelmutant egg chambers were fused to each other. Approximately 1.1% of mael egg chambers showed <15 nurse
cells and 15 ring canals with or without an oocyte,
indicating that these abnormal egg chambers with reduced cell numbers are generated by the arrest of cystoblast mitotic divisions (Fig. 6C [panels c,d], D [panel a]).
About 1.3% of egg chambers showed a compound phenotype that contains <30 nurse cells and two oocyte-like
nuclei with two distinct populations of ring canals (Fig.
6C [panel e], D [panel b]). The compound phenotype was
generated by fusion of egg chambers and defective cystocyte division. These phenotypes are similar to thoseobserved in gTub mutant ovaries (Tavosanis and Gonzalez2003). We thus conclude that mael is required for bothgerm and somatic follicle cell development, possibly bycoordinating components of the MTOC, such as gTub, andthereby regulating proper MT organization.
Mutation in mael triggers Chk2-independent defects
in MT polarity
Most Drosophila piRNA pathway mutations lead to germline DNA damage and disrupt axis specification throughactivation of Chk2, which functions in DNA damage
signaling (Cook et al. 2004; Chen et al. 2007; Pane et al.
2007). The axis specification defects of the oocyte that arelinked to any of several piRNA pathway mutations are
genetically suppressed by a loss of function mutation in
mnk, which encodes a Chk2 homolog required for DNA
damage signaling (Hari et al. 1995; Brodsky et al. 2004).
Defects in MT organization in the mael oocyte may besecondary to DNA damage due to defects in the piRNA
pathway. To test this, we generated double mutants withmnk and analyzed the localization of axis determinants
Osk and Grk. Loss of mael function disrupted theposterior localization of Osk and the dorsal anterior
localization of Grk in the oocyte, and the localization ofboth proteins was not restored in mnk;mael double
mutants (Fig. 7). Thus, the axis specification defect of maeloocytes does not appear to be triggered by germline-specific
DNA breaks and the activation of damage signaling
through Chk2.
Discussion
In this study, we showed that Mael is an MTOC component and that dynamic organization of MTs does not occur
in developing mael oocytes, which correlates withmislocalization of other MTOC components. We also
observed that loss of mael affects the number and positionof the oocytes in egg chambers and induces fusion of egg
chambers. These results indicate that Mael specificallyregulates MTOC formation, and thereby plays a key role in

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Figure 7. mnk does not suppress the mael phenotype. (A)Mutations in mnk do not suppress the Osk localization defectin mael mutant ovaries. (Top) In the control oocyte (stage 9), Osk
is localized at the posterior region of the oocyte (arrow). (Middle)
In mael egg chambers, the localization of Osk was dispersed
throughout the oocyte. (Bottom) In mnk;mael double mutant egg
chambers, the mislocalization of Osk was not rescued. (B) In thecontrol, Grk is localized at the dorsal anterior cortex (arrow) near
the oocyte nucleus (asterisk). (Bottom) In mnk;mael double
mutant egg chambers, the localizations of Osk and Grk are also
dispersed throughout the oocyte. (Middle) In mael egg chambers,
Grk was not accumulated. (Bottom) In mnk;mael double mutant
egg chambers, the mislocalization of Grk was not rescued. DAPI
(blue) marks the cell nuclei. Bars, 20 mm.

coordinating dynamic MT organization during Drosophila
oogenesis.
Initial polarization of the oocyte during the oocyte
specification phase in the germarium requires replacementof the fusome by a polarized MT network, which correlates with the formation of the MTOC (Lin et al. 1994; Linand Spradling 1995; Grieder et al. 2000). Mael is concentrated in the centrosomal region and is colocalized withaTub and gTub during cyst cell divisions. gTub does not
migrate to a developing oocyte in mael germariums,
suggesting that Mael is required for the migration ofcentrioles from the cytoplasm of cysts to pro-oocytes
in the germarium. Currently, the detailed mechanism
by which Mael functions in MT organization is not clear.
The simplest hypothesis is that Mael might serve asa platform that nucleates other MTOC components toform a functional MTOC. A previous report has shownthat weaker mutant alleles of gTub affect the number ofnurse cells and oocytes within the egg chamber (Tavosanis
and Gonzalez 2003). These gTub mutant defects are very
similar to those found in mael mutants in this study. gTub
is involved in the nucleation of MTs and is present in thecentrosomes and MTOCs in many different systems (Jengand Stearns 1999; Moritz and Agard 2001). It was hypothesized that reduced activity of gTub could activate theoocyte determination program in one of the nurse cellsby ectopically presenting MTOC material (Tavosanis andGonzalez 2003). Our findings that gTub does not accumulate at centrosomes in the mael germarium and isectopically expressed in the mael egg chamber suggestthat Mael regulates localization of gTub at centrosomesDownloaded from genesdev.cshlp.org on November 15, 2011 - Published by Cold Spring Harbor Laboratory Press

Mael coordinates MT organization

through its complex formation and is thereby involved inproperly organizing or positioning the MTOC.
PIWI proteins function in transposon silencing via association with piRNAs and maintain genome integrity
during germline development. Recent studies have suggested that PIWI proteins in sea urchin (Seawi) and
Xenopus (Xiwi) can interact with the MTs of the meiotic
spindle (Rodriguez et al. 2005; Lau et al. 2009), while fly
ovarioles with mutations in any of several piRNA pathway
genes, including spn-E, aub, and armi, have disorganizedMTs (Chen et al. 2007; Klattenhoff et al. 2007; Pane et al.
2007). This raises the possibility for either a functional roleof PIWI proteins in the machinery that impacts on MT
organization (in addition to transposon silencing), a role ofthe MT cytoskeleton in piRNA generation, or both. Ourfindings further corroborate a link between componentsof the piRNA pathway and proper MT organization.
Although we found that Mael forms a complex with
MTOC components, we were unable to identify components of the piRNA pathway in this complex. This is incontrast to observations of the mouse Mael homolog,
which functions in the piRNA pathway (similar to fly
Mael) and interacts with mouse PIWI proteins in the testes(Costa et al. 2006). Mouse Mael in the testes is almost
exclusively cytoplasmic with accumulation at nuage(Soper et al. 2008). In contrast, fly Mael is located in boththe nucleus and the cytoplasm in the ovary and is known
to shuttle between them. Thus, one possibility is that in
fly ovaries, there may exist nuclear Mael complexes involved in both piRNA generation and transposon silencing,which are distinct from the cytoplasmic complex containing MTOC components that we identified in this study.
Female flies with mutations in several genes in thepiRNA pathway often lay eggs with axis patterning defects
because of MT cytoskeletal changes that result in the
mislocalization of bic, grk, and osk mRNAs within the egg
chamber (Chen et al. 2007; Klattenhoff et al. 2007; Paneet al. 2007). These defects have been linked to the Chk2
DNA damage checkpoint that may be activated by increased retrotransposon transcript levels in mutants defective in piRNA biogenesis. However, because a mutationin mnk does not suppress the mislocalization of Osk andGrk in the mael oocyte, the axis specification defect ofmael oocytes does not appear to be triggered by theactivation of germline-specific DNA breaks and damage signaling through Chk2. In addition, a mutationin the mei-W68 locus, which encodes the Drosophila
Spo11 homolog and induces meiotic double-strand
breaks in chromosomes (McKim and Hayashi-Hagihara
1998), cannot suppress the axis specification defect of maeloocytes (Findley et al. 2003). Therefore, these results
suggest that the axis specification defects of mael oocytes
are not a secondary consequence of DNA damage signaling. However, it has been shown that in mael mutant
ovaries, Vas is post-translationally modified (Findley et al.
2003), which is another hallmark of checkpoint activation (Ghabrial and Schupbach 1999). These results
together imply that, acting not only through Chk2, thefunctions of Mael in MT organization are in parallel with
its function in piRNA generation and transposon silencing.

There are mutants—including zuc and spn-E, which are
piRNA pathway genes—whose axis defects cannot be
rescued by mnk mutations (Pane et al. 2007). Vas also
appears modified in these mutants, although the relationship with activated checkpoint-modified Vas is unclear(Pane et al. 2007).
Given that Mael is a new component of the MTOC in
the Drosophila ovary, identification of a domain within
Mael that is responsible for binding to other MTOC
components could aid in understanding how Mael nucleates and regulates MTOC formation. Because Maelcontains an evolutionarily highly conserved domain of
unknown function, termed the Mael domain (Zhang
et al. 2008), determination of its crystal structure should
prove valuable in elucidating mechanisms of both MTOC
formation and piRNA generation processes.
Materials and methods
Drosophila strains
For ovary staining and molecular work, y w or maelM391/TM3
was used as a control. The mutant alleles and allelic combinations
used in this work were maelM391/Df(3L)BSC554 (described as
maelM391/Df) and cnnHK21. maelM391/TM3 Sb (Clegg et al. 1997;
Findley et al. 2003) and mnkP6/CyO were kind gifts from T. Kai(National University of Singapore), and w1118; Df(3L)BSC554/
TM6C, Sb1 and cn1 cnnHK21 bw1/CyO, l(2)DTS5131 were obtained from the Bloomington Drosophila Stock Center at Indiana
University.
Production of an anti-Mael monoclonal antibody
An anti-Mael monoclonal antibody was raised specifically against
the N terminus of the protein. A 200-amino-acid N-terminal
fragment of Mael, fused with glutathione S-transferase (GST),
was used as the antigen to immunize mice. The anti-Mael monoclonal antibody was produced essentially as described previously(Ishizuka et al. 2002) and was purified from culture supernatantof hybridoma cells under standard procedures using ThiophilicSuperflow resin (BD Biosciences).
Immunoprecipitation
Ovaries were dissected manually from adult flies in cold phosphatebuffered saline (PBS) and homogenized in 300 mL of Digitonin-based
binding buffer containing 30 mM HEPES-KOH (pH7.3), 150 mM
potassium acetate (KOAc), 4 mM magnesium acetate (MgOAc),
1 mM dithiothreitol (DTT), 50 ng/mL Digitonin, 2 mg/mL
pepstatin, 2 mg/mL leupeptin, and 0.5% aprotinin. In Supplemental
Figure S2E, immunoprecipitation was performed using Empigenbased binding buffer containing 1% Empigen BB (Sigma), 1 mM
EDTA, 1 mM DTT, 2 mg/mL pepstatin, 2 mg/mL leupeptin, and
0.5% aprotinin in PBS. After centrifugation at 13,500 rpm for 1
min at 4°C, the supernatant was collected into a new microcentrifuge tube and kept on ice. The pellet was rehomogenized in
300 mL of binding buffer and recentrifuged as described above.
The second aliquot of supernatant was combined with the first.
These steps were repeated three times. Collected supernatant
was used as the sample solution for immunoprecipitation. The
protein concentration of the sample solution was adjusted eachtime to 5 mg/mL with binding buffer, and incubated with 5 mg of
purified anti-Mael monoclonal antibody immobilized on GammaBind beads (Amersham Bioscience) for 2 h at 4°C with rotation.
After incubation, the bead fractions were extensively washed at

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Sato et al.

least five times using binding buffer, and the protein complexes
were eluted from the beads with sample buffer containing SDS byheating for 10 min at 70°C and then loaded onto SDS-PAGE gels.
After electrophoresis, proteins were visualized by silver staining
using Silver-Quest (Invitrogen) or processed for Western blot
analyses. Protein bands detected by silver staining were excisedfor mass spectrometric analysis.
Western blot analysis
Whole embryo, larval, and pupal bodies were collected and homogenized in lysis buffer containing 30 mM HEPES-KOH (pH 7.3),
150 mM KOAc, 4 mM MgOAc, 1 mM DTT, 50 ng/mL digitonin,
2 mg/mL pepstatin, 2 mg/mL leupeptin, and 0.5% aprotinin. Aftercentrifugation at 13,500 rpm for 10 min at 4°C, the supernatants
were used as samples. Ovary lysates were prepared as describedabove. Western blot analysis was performed as described previously (Miyoshi et al. 2005). Culture supernatant of hybridoma
cells producing anti-Mael monoclonal antibody was used without
dilution as a primary antibody. Anti-Cnn was a kind gift from T.C.
Kaufman (Indiana University) and used at 1:2000. Anti-D-TACC
was a kind gift from J.W. Raff (University of Oxford) and used at
1:10,000. Anti-Msps was a kind gift from H. Ohkura (University of
Edinburgh) and used at 1:10,000 dilution. Anti-gTub (GTU88;
Sigma, #T6557) was used at 1:2000. Anti-Ago1, anti-Ago2, antiAgo3, anti-Piwi, and anti-Aub antibodies have been described
previously (Miyoshi et al. 2005; Gunawardane et al. 2007; Saito
et al. 2006; Nishida et al. 2007). Anti-aTub was obtained from theDevelopmental Studies Hybridoma Bank and used at 1:1000.
Immunohistochemistry

GCTCCTAAGAAGCATAGT-39 and 59-GGGCTCGAGTTATTTTTTAAGTTTCCCATC-39. PCR products were digested with
BamHI and XhoI, and then subcloned into pBlueScript SK. The
entire inserted sequence was amplified by PCR using the resultant
plasmid as a template with T7 (59-TAATACGACTCACTATAG
GG-39) and T3 (59-ATTAACCCTCACTAAAGGGAA-39) promoter
primers. cnn, cdc27 cDNAs were amplified by RT–PCR from
Drosophila ovarian total RNA using the following specific primers:for cnn, 59-TAATACGACTCACTATAGGGCAGATGGCGG-39
and 59-TAATACGACTCACTATAGGGCTTCTCC TCC-39; forcdc27, 59-TAATACGACTCACTATAGGGGCCGAGGACA-39
and 59-TAATACGACTCACTATAGGGCTTCATTGCA-39. It
should be noted that the 59 end of each primer contained the T7
RNA polymerase promoter sequence (59-TAATACGACTCACTATAG-39). PCR products were purified using the QiaQuick gel
extraction kit (Qiagen) according to the manufacturer’s instructions. Purified PCR products were used to produce dsRNAs using
Megascript T7 and T3 transcription kits (Ambion). The resultant
RNAs were purified according to the manufacturer’s instruction,
heated for 5 min at 95°C, and then left to cool to room
temperature. S2 cells were grown at 26°C in Schneider’s Insect
medium (Sigma) supplemented with 10% fetal calf serum (Gibco),
50 mg/mL streptomycin, and 50 mg/mL penicillin. For RNAi in S2
cells, ;5 3 106 cells were suspended with 5 mg of dsRNA andincubated for 4 d at 26°C.
Mitotic index
To determine the mitotic index, S2 cells were processed for
immunofluorescence microscopy as described above. The cells
were labeled with anti-pH3, as a marker of mitotic cells and withDAPI to visualize DNA (Goshima et al. 2007). The percentage of
mitotic cells was determined using the image processing software
ImageJ.

Ovaries were dissected manually from adult flies in cold PBS. For
MT detection, dissected ovaries were fixed immediately in cold
methanol for 10 min at 20°C, rehydrated in PBS for 15 min,permeabilized with 0.5% NP-40 in PBS containing 0.1% Tween
20 and 2% bovine serum albumin (BSA) for 30 min, and thenincubated overnight at 4°C with anti-aTub antibodies. For
detection of aTub, anti-aTub antibody (rat YL1/2; Serotec) and
FITC-conjugated anti-aTub (Sigma, #F2168) were used at a 1:150
dilution in Figures 2–4 and at a 1:50 dilution in Figure 6. Anti-Cnn
and anti-gTub (Sigma, #T3559) antibodies were used at 1:500 and
1:100 dilutions, respectively. Anti-Orb, anti-Grk, and anti-DE-cad
antibodies were obtained from the Developmental Studies
Hybridoma Bank and used at 1:400, 1:40, and 1:20 dilutions,
respectively. Anti-Osk and anti-Vas were a kind gift from A.
Nakamura (RIKEN, CDB) and used at a 1:3000 and 1:1000
dilutions, respectively. PY20 (BD Transduction Laboratories) andanti-pH3 (Upstate Biotechnologies) antibodies were used at 1:100
and 1:1000 dilutions, respectively. Alexa Fluor488-conjugatedanti-mouse IgG, Alexa Fluor488-conjugated anti-mouse IgG2a,
Alexa Fluor546-conjugated anti-mouse IgG2b, Alexa Fluor633conjugated anti-mouse IgG, Alexa Fluor546-conjugated anti-rabbit
IgG, Alexa-Fluor488 conjugated anti-guinea pig IgG, and Alexa
Fluor488-conjugated anti-rat IgG (Molecular Probes) were used as
secondary antibodies. Ovaries were mounted in VectaShield withDAPI (49,6-diamidino-2-phenylindole dihydrochloride) (Vector
Laboratories). All images were collected using a Zeiss LSM510
laser-scanning microscope. Image processing and annotation
were performed using Adobe Photoshop (Adobe) and ImageJ(NIH) software.

Aequorea coerulescens GFP (AcGFP) cDNA was amplified by
PCR from pAcGFP1 vector (Clontech) using the primers 59-TTT
TTCCATGGTGAGCAAGGGCGCCG-39 and 59-GATACATTC
ACGTGCTAGAATTCCTGCCTTGTACAGC-39. aTubulin84B(aTub) cDNA was amplified by RT–PCR from Drosophila ovarian
total RNA using the primers 59-GCTGTACAAGGCAGGAATTCTAGCACGTGAATGTATC-39 and 59-TTTTTGCTAGCTTAGTACTCCTCAGCGCCCT-39. A cDNA encoding an AcGFP-aTubfusion protein was then amplified by an ‘‘overlap PCR’’ method
using AcGFP and aTub PCR products with primers 59-TTT
TTCCATGGTGAGCAAGGGCGCCG-39 and 59-TTTTTGCTAGCTTAGTACTCCTCAGCGCCCT-39. The PCR product wasdigested with NcoI and NheI, and then subcloned into a vector
downstream from the metallothionein promoter. The AcGFPaTub-expressing plasmid vector was transfected into S2 cells
using Cellfectin (Invitrogen) according to the manufacturer’s
instructions. The AcGFP-aTub fusion protein was induced byadding CuSO4 to the culture medium (final concentration ;0.5mM). For time-lapse video fluorescence microscopy imaging,
AcGFP-aTub trnsfected S2 cells were plated in poly-L-lysinecoated glass-bottom culture dishes (Matsunami) on day 4 after
RNAi treatment. All mages were collected in 35- to 55-sec
intervals using a Zeiss LSM710 laser-scanning microscope.
Image processing and annotation were performed using ZEN
software (Carl Zeiss).

Cell culture and RNAi

Acknowledgments

mael cDNA was amplified by RT–PCR from Drosophila ovarian
total RNA using the following primers: 59-GGGGGATCCATG

We are grateful to G. Goshima, T. Kai, T. Kaufman, H. Kose, A.
Nakamura, H. Ohkura, and J.W. Raff for providing antibodies, fly

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Live-cell imaging

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Mael coordinates MT organization

stocks, protocols, and advice. Special thanks to I. Sagawa for mass
spectrometry. We also thank members of the Siomi laboratory fordiscussions and helpful comments on the manuscript. This work
was supported by MEXT (Ministry of Education, Culture, Sports,
Science, and Technology of Japan) grants to H.S., M.C.S., M.K.N.,
and K.S. M.C.S. is supported by CREST (Core Research for
Evolutional Science and Technology) from JST (Japan Scienceand Technology Agency).

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