Research Article

3649

COPI-mediated membrane trafficking is required forcytokinesis in Drosophila male meiotic divisions
Daishi Kitazawa, Masamitsu Yamaguchi, Hajime Mori and Yoshihiro H. Inoue*
Insect Biomedical Research Center, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585,
Japan*Author for correspondence (yhinoue@kit.ac.jp)

Journal of Cell Science

Accepted 10 April 2012
Journal of Cell Science 125, 3649–3660
ß 2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.103317

Summary
The coatomer protein complex, COPI, mediates retrograde vesicle transport from the Golgi apparatus to the ER. Here, we investigatedthe meiotic phenotype of Drosophila melanogaster spermatocytes expressing dsRNA of 52 genes encoding membrane-traffickingrelated factors. We identified COPI as an essential factor for male meiosis. In Drosophila male meiotic divisions, COPI is localized in
the ER–Golgi intermediate compartment of tER–Golgi units scattered throughout the spermatocyte cytoplasm. Prior to chromosome
segregation, the vesicles assemble at the spindle pole periphery through a poleward movement, mediated by minus-end motor dynein
along astral microtubules. At the end of each meiotic division, COPI-containing vesicles are equally partitioned between two daughter
cells. Our present data strongly suggest that spermatocytes possess a regulatory mechanism for equal inheritance of several types ofmembrane vesicles. Using testis-specific knockdown of COPI subunits or the small GTPase Arf or mutations of the cCOP gene, we
examined the role of COPI in male meiosis. COPI depletion resulted in the failure of cytokinesis, through disrupted accumulation of
essential proteins and lipid components at the cleavage furrow region. Furthermore, it caused a reduction in the number of overlapping
central spindle microtubules, which are essential for cytokinesis. Drosophila spermatocytes construct ER-based intracellular structuresassociated with astral and spindle microtubules. COPI depletion resulted in severe disruption of these ER-based structures. Thus, we
propose that COPI plays an important role in Drosophila male meiosis, not only through vesicle transport to the cleavage furrow region,
but also through the formation of ER-based structures.
Key words: Cell division, COPI, Cytokinesis, Drosophila melanogaster, Meiosis

Introduction
Cytokinesis is the final step of cell division, and results from theconstriction of a contractile ring comprising F-actin and myosin
fibers (Satterwhite and Pollard, 1992). Microtubule bundles known
as the central spindle help position the actomyosin ring at theequator (Glotzer, 2001; Cao and Wang, 1996; Inoue et al., 2004).
A protein complex called centralspindlin plays a key role ininitiating contractile ring formation (D’Avino et al., 2005;
Mishima and Glotzer, 2003). Completion of cytokinesis isdependent not only on contractile ring formation, but also on
vesicle transport (Glotzer, 2001; Albertson et al., 2005; Montagnac
et al., 2008; Prekeris and Gould, 2008).
The delivery of membrane components to the cleavage furrow
region of the equatorial plasma membrane is thought to involvetwo different vesicle transport pathways: the secretory pathway
and the endocytic pathway. The Drosophila melanogastersecretory pathway involves several transport proteins. Syntaxin 5(one of the SNAREs required for membrane fusion) is necessary
for male meiotic divisions (Xu et al., 2002). Lva (a Drosophila
ortholog of golgin) is essential for cellularization, which is a
cognate cytokinesis process in early embryos (Sisson et al., 2000).
An fws-encoded Drosophila Cog5 plays a role in intra-Golgi
vesicle transport, and is essential for cytokinesis in male meiotic
divisions (Farkas et al., 2003). Using proteomic analysis, Skop andcolleagues revealed that one-third of proteins accumulated in the
mammalian midbody were secretory or membrane-trafficking

proteins. RNAi experiments in Caenorhabditis elegans confirmed
the dependence of cytokinesis on these proteins (Skop et al., 2004).
The endocytic pathway involves intracellular transport throughearly and recycling endosomes (Boucrot and Kirchhausen, 2007).
The small GTPase, Rab11, is important in regulating membranetrafficking through recycling endosomes (Giansanti et al., 2007;
Prekeris and Gould, 2008). Giansanti and colleagues reported theaccumulation of Golgi-derived vesicles at the midzone in rab11
mutant spermatocytes, and the perturbation of membrane vesicle
insertion into the plasma membrane. Recruitment of Rab11 to thecleavage furrow is dependent on the regulatory factors, Giotto
and Fwd (Giansanti et al., 2007). Nuf, a Rab11 effector, isrequired for targeting of endosomal vesicles and F-actin throughthe central spindle and furrow microtubules (Albertson et al.,2008; Cao et al., 2008). Furthermore, in cell abscission, theRab11 interacting proteins, Fip3/Nuf and Cep55, are responsible
for targeting of recycling endosomes to the midbody (Simon
et al., 2008; Zhao et al., 2006). These recycling endosomes arerich in Arf6 GTPase, which interacts with Rab11-mediatedmembrane addition, but is not required for central spindle
formation, contractile ring assembly, or targeting of recycling
endosomes to the central spindle (Dyer et al., 2007). The
Drosophila bru gene encodes TRAPP (membrane-trafficking
transport protein particle) II complex, which is required for
Rab11 localization at the cleavage furrow, and ingression incytokinesis (Robinett et al., 2009).

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To identify other cytokinesis factors, two independent large
RNAi screens were previously performed in S2 cultured cells(Echard et al., 2004; Eggert et al., 2004). Several subunits of a
coatomer protein complex, COPI, were commonly identified.
The first screen identified five out of seven subunits as essential
cytokinesis factors. Consistent with these results, brefeldin A(which inhibits coatomer assembly) disrupted the completion ofcytokinesis (Sisson et al., 2000; Skop et al., 2004).
Transported vesicles are coated with one of three types of
coatomer protein complex (COPI, COPII, or clathrin), according
to cellular transport steps. COPI is a protein complex consistingof seven subunits (a, b, b9, c, d, e, and f). COPI-containing
vesicles return membrane components and selected proteins fromthe cis end of the Golgi apparatus to the ER (Kondylis andRabouille, 2009). In budding yeast, all COPI subunits except forf were shown to be essential genes. The seven subunits arereported to be ubiquitously expressed in somatic and germline
cells during Drosophila development (Grieder et al., 2005).
Mutants for cCOP or dCOP were lethal at the embryonic stages.
The cCOP mutant displayed marked morphogenetic defects ofthe epithelia and tracheal tubes (Grieder et al., 2008; Jayaram
et al., 2008). Immunolocalization of the bCOP subunit in rat cells
by electron microscopy revealed the localization of COPI overthe Golgi apparatus, mainly at the cis side (Oprins et al., 1993). In
Drosophila cells, bCOP and cCOP colocalize with the Golgi
stacks (Jayaram et al., 2008; Ripoche et al., 1994). COPI is
involved in the formation and/or maintenance of the Golgi
apparatus (Kamena et al., 2008; Lippincott-Schwartz et al.,1998). A large-scale RNAi screen revealed that COPI is involved
not only in retrograde vesicle transport, but also in the regulationof lipid homeostasis (Beller et al., 2008). The screen identified
several genes encoding subunits of COPI, but not of COPII orclathrin, as essential regulators of lipid droplets. These lipid
droplets are ubiquitous lipid storage organelles, and may be
important suppliers of vesicle components (Cermelli et al., 2006).
Thus, COPI may mediate the transport of lipid droplets out of theinternal stores. Mutants for the COG family, which regulates
COPI-mediated transport through the Golgi cisternaes,
demonstrated cytokinesis defects (Farkas et al., 2003). Thus, itappears that COPI may be involved in cytokinesis. However, its
precise role remains unclear. In the present study, we investigatedthe cytokinesis role of COPI in Drosophila male meiosis.
The Drosophila spermatocyte has frequently been used tocharacterize vesicle transport-related factors in cytokinesis(Fuller, 1993). The cell is the largest diploid cell to undergo
cell division. A cyst, which is a unit of 16 spermatocytes,
simultaneously gives rise to 64 spermatids following two
successive meiotic divisions. Each onion-stage spermatid in a
cyst contains an equal-sized nucleus and mitochondrial-derived
Nebenkern, achieved through correct chromosome segregationand cytokinesis in germline cells, and by equal partitioning of
mitochondria (Fuller, 1993; Maines and Wasserman, 1997).
Phase-contrast microscopy facilitates the identification of subtle
defects in meiotic divisions (Castrillon et al., 1993; Ichihara et al.,2007). The Drosophila spermatocyte possesses well-developed
membranous cellular structures, and therefore constitutes asuitable model for investigating the involvement of membranetrafficking in cell divisions (Bobinnec et al., 2003). Primary
spermatocytes posses particularly long astral microtubules(Fuller, 1993; Inoue et al., 2004; Inoue et al., 2011). By
contrast, the spindle microtubules form within a nuclear space,surrounded by a multilayer of parafusorial membranes (Fuller,
1993; Tates, 1971). Thus, it is easy to distinguish spindle
microtubules from asters. Inoue and colleagues reported that a
microtubule-associated protein, Orbit/CLASP, accumulated on
spindle microtubules, but not on asters (Inoue et al., 2004). Thus,
it appears that each type of microtubule has a different
biochemical feature.
In the present study, we initially performed an RNAi screen to
identify essential genes for male meiosis among genes encoding
membrane trafficking-related factors. We identified COPI
subunits and regulatory proteins as essential cytokinesis factors.
Next, we examined the dynamics of COPI-containing vesicles
during the progression of cell division in Drosophila malemeiosis. Prior to chromosome segregation, the vesicles assembleat the spindle pole periphery through a poleward movement,
mediated by minus-ended motor dynein along astral
microtubules. At the end of each meiotic division, the vesicles
are equally partitioned between two daughter cells. We revealed
that COPI depletion resulted in the failure of cytokinesis, throughdisrupted accumulation of essential proteins and lipid
components at the cleavage furrow. Furthermore, it caused a
reduction in the number of overlapping central spindle
microtubules, which are essential for cytokinesis. Drosophila
spermatocytes construct ER-based intracellular structuresassociated with astral and spindle microtubules. COPI depletion
resulted in severe disruption of these ER-based structures. Thus,
we believe that COPI plays an important role in male meiosis, notonly through vesicle transport to the cleavage furrow region, butalso via the formation of intracellular structures.
Results
Identification of COPI subunits as essential factors for
Drosophila male meiosis

Whole genome screen of Drosophila S2 cells previously identified
genes encoding certain subunits of the COPI complex as essential
for cytokinesis (Echard et al., 2004; Eggert et al., 2004). Here, weinvestigated the meiotic phenotype of Drosophila spermatocytes,
by performing knockdown of 52 genes for membrane traffickingrelated factors (including COPI subunits). Using the Drosophilatargeted gene expression system, Gal4/UAS, we performed
spermatocyte-specific knockdown of genes for the seven COPI
subunits, five arf genes encoding COPI assembly factors(supplementary material Table S1), and 40 membrane traffickingrelated genes. For induction of dsRNA for these genes, we mainly
used bam–Gal4 driver and UAS-RNAi stocks from the Vienna
Drosophila RNAi Center. We observed that each wild-type,onion-stage spermatid contained an equal-sized nucleus andmitochondrial-derived Nebenkern (Fig. 1A,B). Knockdown malesof COPI a, b, c, and f subunits exhibited abnormal spermatids, witha higher ratio of nucleus to Nebenkern (Fig. 1A,C,D). Our genetic
data indicate the incorrect execution of cytokinesis. Spermatidsfrom knockdown males of all four subunits exhibited similar
meiotic phenotypes.
To confirm our observations, we examined male meiotic
phenotypes of mutants for the cCOP subunit (Fig. 1A,E).
Hypomorphic cCOP mutants died before the third instar larvalstage, because of the viability requirement for COP. We therefore
provided cCOP expression by using an arm-Gal4 driver, which
induces ubiquitous UAS-dependent gene expression in somatic
cells rather than in germline cells (Kirchner et al., 2008). We
generated arm-Gal4/+;cCop{XP}d06498 flies, with an XP element

Drosophila male meiosis requires COPI

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Furthermore, testis-specific knockdown of five Arf1 family
members revealed the most severe cytokinesis phenotype inonion-stage spermatids (Fig. 1F, 98%; n5820) from arf72A
knockdown males containing multiple normal-sized nuclei. In
arf79F knockdown males, cytokinesis defects were also observed
repeatedly, but less frequently (12%, n52412). Treatment of
meiotic cells with the Arf1 inhibitors, brefeldin A or Exo1,
influenced cytokinesis in meiotic divisions (Fig. 1F). Taken
together, our results indicate that the COPI complex assembled
by Arf1 is required for cytokinesis in Drosophila male meiotic
divisions. We raised antibody against aCOP and confirmed that itrecognizes single polypeptides in testis extracts, which increasedin amount after overexpression of aCOP and significantly
reduced by expression of its dsRNA (Fig. 1G).

Journal of Cell Science

COPI localization in an ER–Golgi intermediate compartment

Fig. 1. Loss of COPI subunits or inhibition of COPI assembly results in the
failure of cytokinesis in Drosophila male meiotic divisions.
(A) Quantification of cytokinesis defects in onion-stage spermatids fromcontrol and bam–Gal4 males, knockdown males for COPI subunits and cCopmutant males with the genotype arm-Gal4;ccop{XP}d06498/P{SUPor-P}G06383.
Testes from 15 males of each genotype were examined. (B–E) Phase-contrast
micrographs of onion-stage spermatids derived from control males (B), aCopknockdown males (C), cCop knockdown males (D) and cCop mutant maleswith the genotype arm-Gal4;ccop{XP}d06498/P{SUPor-P}G06383 (E) Scale bar:
10 mm. (F) Effects of two Arf1 inhibitors, brefeldin A (BFA) and Exo1, on
wild-type spermatids. Cells underwent meiotic divisions in the presence orabsence of the inhibitors. Spermatids from arf72A knockdown male are also
shown. (G) Western blots of the Drosophila aCOP subunit. The antibody
against aCOP recognized a single polypeptide of 138 kDa, which corresponded
to the predicted molecular mass of aCOP in western blot analysis using testis
extracts prepared from wild-type adult males. Overexpression of the aCop genein hsp-Gal4.UAS-aCop males resulted in accumulation of the polypeptide.
Conversely, extracts prepared from bam-Gal4.UAS-aCop IR males expressing
aCop dsRNA had less polypeptide.

integrated into the cCop locus. Somatic cells transcribed cCopmRNA from the XP element, in a manner that was dependent on
arm-Gal4, whereas germ cells transcribed cCop mRNA lessabundantly. The arm-Gal4/+;cCop{XP}d06498 males displayed
meiotic defects in cytokinesis at a low frequency (Table 1). We
used a different insertional mutation, induced by a UAS-lacking
P element, P{SUPor-P}, as one of the cCop alleles (Table 1).
This mutation increased the frequency of abnormal cells
exhibiting cytokinesis phenotypes. Our genetic data from testisspecific knockdown of COPI subunits, and the male meiotic
phenotype of cCop mutants, consistently indicated therequirement of COPI for cytokinesis.

Prior to further investigation of the biological role of COPI in malemeiosis, we investigated the cellular distribution of the COPI
complex in primary spermatocytes. Immunostaining of premeiotic
spermatocytes with antibody against aCOP showed that theantibody recognized 20–40 foci in the cells (supplementarymaterial Fig. S1). Moreover, aCOP knockdown spermatocytes
exhibited considerably fewer foci (42% of normal control, n551)
and reduced whole signal intensity (supplementary material Fig.
S2). The aCOP foci perfectly overlapped with the mRFP–cCOP
foci in spermatocytes (Fig. 2A). Thus, it is likely that Drosophila
aCOP forms part of the COPI complex with additional subunits, as
in other organisms (Hosobuchi et al., 1992; Duden et al., 1998).
Hereafter, we consider foci stained with aCOP antibody to indicate
the presence of COPI.
In mammalian cultured cells, COPI co-localizes with the Golgi
apparatus (Griffiths et al., 1995). We therefore examined whether
Drosophila COPI continuously co-localizes with several Golgi
markers in premeiotic spermatocytes. In Drosophila, as in yeast
or worms, the Golgi apparatus exists as multiple stacks in thecytoplasm. In premeiotic spermatocytes, these Golgi stacks
appeared as 20–40 horseshoe-shaped foci (Inoue et al., 2011).
We investigated the co-localization of COPI by using several
Golgi markers: GM130 for the cis side and Rab11 for the transside of the Golgi stacks; KDEL for the ER–Golgi intermediate
compartment (ERGIC); and Sec31 for the transitional ER (tER)sites (Fig. 2B). We observed that the COPI distribution perfectly
overlapped with the ERGIC marker in premeiotic spermatocytes,
but was not identical to foci visualized using the other Golgi
markers (Fig. 2C–F). Given that COPI may play an importantrole in vesicle transport between the ER and Golgi membranes, it
is likely that most of the COPI is distributed in the ERGIC of theGolgi stacks.
Astral microtubules are required for equal partitioning of
COPI-containing vesicles

Using the aCOP antibody (Fig. 1G), we examined the cellular
localization of COPI-containing vesicles during male meiotic
divisions (supplementary material Fig. S3). We observed thatspermatocytes entering meiosis contained a twofold higher numberof COPI vesicles than did premeiotic spermatocytes (supplementarymaterial Figs S1, S3). Moreover, each vesicle diminished in size,
implying their fragmentation into smaller pieces during prophase toprometaphase. At each meiotic division, the vesicles were equallypartitioned between two daughter cells (supplementary material Figs
S3, S4). At prophase, they were uniformly distributed throughout

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Table 1. Quantification of meiotic defects in spermatids from cCOP mutant males with somatic rescue induced by arm–Gal4
Nebenkern-to-nuclei ratio (% cells)
Genotype
arm-GAL4
cCop{XP}d06498/+*
arm-Gal4/+;cCop{XP}d06498
arm-Gal4/+;cCop{XP}d06498/P{SUPor-P}cCopKG06383

AbnormalNo. of onion-stage
spermatids scored

Normal

1:0

1:2

1:3

1:4

1:.5

Total

Macro/micronuclei (% cells)

2820
2721
821
362

100.0
100.0
99.5
65.2

0.0
0.0
0.0
2.2

0.0
0.0
0.5
6.9

0.0
0.0
0.0
3.0

0.0
0.0
0.0
5.5

0.0
0.0
0.0
17.1

0.0
0.0
0.5
34.8

0.0
0.0
2.6
3.6

Journal of Cell Science*cCop{XP}d06498 homozygotes are lethal.
Testes from 15 males were scored per genotype.

the cytoplasm. Prior to chromosome segregation, they wereassembled into two groups, each containing similar numbers ofvesicles. The vesicles were localized on astral microtubules, andbecame comprehensively assembled at the spindle pole periphery
prior to metaphase. It appears that the vesicles are kept away fromthe midzone, in order to achieve equal partitioning between two
daughter cells. During cytokinesis, they are redistributed throughout
the cytoplasm.
During the second meiotic division, we observed the same
distribution of vesicles. On completion of the meiotic divisions,the COPI vesicles in each spermatid were assembled into a single
spherical structure, called an acroblast (supplementary material
Fig. S3F). Immunostaining of male meiotic cells with cCOP
antibody revealed the same distribution (supplementary material
Fig. S4). This characteristic distribution of COPI vesicles in male
meiotic cells was not observed during mitosis of Drosophila S2cells or female meiotic cells (Fig. 3A). Such a characteristic
COPI distribution seems to be closely associated with astral
microtubules in male meiotic cells.

Drosophila spermatocytes possess particularly long astral
microtubules. By contrast, spindle microtubules form within anuclear space, surrounded by a multilayer of parafusorial
membranes (Fuller, 1993; Tates, 1971) (see supplementarymaterial Figs S3, S5). Thus, it is easy to distinguish spindle
microtubules from asters. We subsequently investigated theinfluence of several mutations affecting microtubule structures,on the distribution of COPI vesicles (Fig. 3B,C). Meiotic cells
from bTub85DD/+ males exhibited disrupted formation of astraland spindle microtubules. The characteristic COPI distribution
was no longer present, and the vesicles were uniformly
distributed throughout the cytoplasm. Our results suggest that
COPI vesicle distribution is dependent on microtubule structures.
Meiotic cells from asl males may lack astral microtubules,
because asl is required for spindle pole formation (Bonaccorsi
et al., 1998). Fig. 3B shows the distribution of COPI vesicles
throughout the cytoplasm of asl mutants, including the central
region of metaphase-I-like cells. By contrast, the orbit7 mutationinfluences central spindles and not astral microtubules (Inoue

Fig. 2. Immunodetection of the COPI complex and simultaneous localization of COPI with tER–Golgi marker proteins. (A) Simultaneous immunostaining
of aCOP and fluorescence of mRFP–cCOP. In the merged image (left panel), aCOP and cCOP are displayed in red and green, respectively. DNA is shown inblue. Colocalization of aCOP with cCOP appears as yellow spots. (B) Schematic presentation of tER–Golgi components and their intracellular localization.
(C–F) Simultaneous immunostaining of premeiotic spermatocytes with the aCOP antibody and tER–Golgi markers at the S5–S6 stage of the growth phase.
Developmental stages of spermatocytes were determined by DAPI staining and phase-contrast observation, according to a previous study (Bonaccorsi et al., 2000).
aCOP immunofluorescence is shown in the middle column, and displayed in red in the left column. Immunofluorescence of the Golgi–tER site marker Sec31(C), ER–Golgi intermediate compartment (ERGIC) marker KDEL (D), cis-side marker GM130 (E), and trans-side marker Rab11 (F) is displayed in the left
column, and visualized in green in the merged images. Scale bars: 10 mm.

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Fig. 3. Distribution of COPI-containing vesicles during male meiosis is affected in flies mutants for astral microtubules rather than for spindle
microtubules. (A) Immunolocalization of COPI vesicles in S2 cultured cells at metaphase and in metaphase I cells from wild-type females. Anti-tubulin
immunostaining is shown in green, anti-aCop immunostaining in red and DNA in blue. Note the random distribution of COPI vesicles throughout the cytoplasm of
dividing cells. (B) Immunofluorescence of the COPI vesicles (red) in metaphase I cells from wild-type and mutant males with abnormal microtubule structures. In
wild-type cells, the COPI vesicles appear to be localized on astral microtubules around two spindle poles. In a metaphase-I-like cell from a bTub85DD/+ male, the
COPI vesicles are distributed as far as the central region of the cell. In a metaphase-like cell from an asl1/asl2 male, with few or incorrectly oriented astral
microtubules, the distribution of COPI vesicles is compromised, and a subset of vesicles remains in the center of the cell. In the metaphase-I-like cell from an
orbit7 male, in which spindle microtubules fail to overlap each other at the plus ends, COPI vesicles accumulate around a spindle pole. Scale bars: 10 mm.
(C) Frequency of cells displaying normal distribution of COPI vesicles at metaphase I (M) or anaphase I (A). Spermatocytes from wild-type [M, 85.1% (n5141);
A, 96.4% (n5111)], bTub85DD/+ [M, 34.4% (n596); A, 54.1% (n5185)], asl1 [M, 33.8% (n5139); A, 32.7% (n587)], asl1/asl2 [M, 12.5% (n596); A, 8.2%(n5107)], and orbit7 [M, 74.3% (n574); A, 81.8% (n5110)].

et al., 2004). Thus, COPI distribution was maintained in an orbit7
metaphase-I-like cell (Fig. 3B). In comparison with the orbit7
mutants, the asl mutants contained markedly fewer cells
displaying normal COPI distribution (Fig. 3C).
Thus, our results indicate that microtubule structures are
essential for the distribution of COPI vesicles during meiosis.
Moreover, the equal partitioning of COPI vesicles is dependent
on well-developed astral microtubules in male meiotic cells,
rather than on spindle microtubules.
Requirement of dynein for COPI vesicle transport along
astral microtubules towards the spindle pole periphery

To understand the inheritance of COPI vesicles during malemeiosis, we investigated the movement of mRFP–cCop-labeled
vesicles in a living spermatocyte by means of a time-lapse
experiment (Fig. 4A; supplementary material Movie 1). The
ubiquitously distributed vesicles moved towards each spindle
pole during prometaphase to metaphase. We observed the
movement of several COPI foci from the plus ends of asters
towards a spindle pole, along the microtubules. [All fociexamined (n550 foci in 26 cells) performed the movement.

Arrowhead in Fig. 4A]. These foci eventually converged aroundthe spindle poles on astral microtubules, until metaphase.
Sisson and colleagues proposed minus-ended motor dynein asa candidate for the transport of COPI vesicles (Sisson et al.,2000). We observed the enrichment of dynein around the spindle
poles (Fig. 4B). Moreover, COPI vesicles were distributed on andover a dynein-accumulated region. In mutants for Dhc64C
encoding the dynein heavy chain, or Dhc64C knockdown
spermatocytes, the characteristic COPI distribution was notpresent (67% abnormality, n521 mutant cells, Fig. 4C).
Furthermore, we observed Lva-containing vesicles, which may
correspond to Golgi stacks (Giansanti et al., 2007; Papoulas et al.,2005), dispersed throughout the cytoplasm. Our results suggestthat dynein is required for the transport of COPI and Lvacontaining vesicles, along astral microtubules towards the spindle
poles.
Defective accumulation of cleavage furrow proteins andlipid droplets in COPI-depleted spermatocytes

To understand the primary cytokinesis defects in COPI-depleted
spermatocytes, we investigated perturbations in the accumulation

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S7). These lipid droplets were localized on the astral membranes
until metaphase (100% accumulation; n510). Fibrous
distribution connecting each astral membrane was evident at
metaphase, indicating that the lipid droplets may be distributed
along spindle microtubules, or associated with ER-based spindleenvelopes. We observed prominent accumulation at the central
region until early anaphase (100% accumulation; n511;
Fig. 5H). It appears that the lipid droplets facilitate de novo
membrane synthesis during subsequent cell elongation at lateanaphase. At telophase, lipid droplets were generally lessabundant, and accumulation on the astral membranes was nolonger present, even in wild-type cells. The droplets were locally
distributed along the edge of the central spindles, and on theinvaginated plasma membrane contacting the central spindles[100% accumulation (n515), closed arrowheads in Fig. 5I;
100% accumulation (n58), supplementary material Fig. S6A].
By contrast, in COPI-depleted cells, we observed no distinct
midzone accumulation during metaphase to anaphase. However,the distribution around the astral membranes remained [71%abnormal accumulation (n517), Fig. 5J; 71% abnormal
accumulation (n517), Fig. 5K]. Plasma membrane ingression
failed to occur effectively; where it did occur, the invaginatedmembrane did not contact lipid droplets on the central spindle[30% without contact (n510), open arrowheads in Fig. 5L; 65%
without contact (n523), supplementary material Fig. S6B].
Taken together, our results indicate that disruption of COPImediated membrane trafficking results in defective accumulationof vesicle components, such as cleavage furrow proteins and lipid
droplets, at the cleavage furrow site.

Fig. 4. Time-lapse observation of COPI vesicle transport along astral
microtubules toward spindle poles, and its disruption by depletion ofdynein heavy chain. (A) Spermatocytes derived from males in which btubulin–GFP and mRFP–cCOP are simultaneously expressed were used to
trace the single COPI foci from early prometaphase I (t50 min) to metaphase
I (t530 min). A single COPI focus indicated by an arrowhead is moving
along astral microtubules in the minus-ended direction. (B,C) Simultaneous
immunostaining of anaphase I cells from wild-type (B) or knockdown males
for dynein heavy chain (C) with aCop antibody (red) and dynein heavy chain
antibody (green in B) or Lva antibody (green in C). DNA is shown in blue.
Scale bars: 10 mm.

of cleavage furrow components. In wild-type cells, we observed
the accumulation of three vesicle transport-related proteins(Fig. 5A–C). Rab11 is highly enriched at the cleavage furrow
region during telophase (92% accumulation; n512) (Giansantiet al., 2007). Nuf, an effector of Rab11, is designated as a
midzone-concentrated protein (91% accumulation; n511)(Polevoy et al., 2009). Clathrin, another coatomer protein
complex, is enriched at the midzone region during telophase[100% accumulation (n510), Fig. 5C]. COPI-depleted cells
failed to accumulate these three proteins at the midzone region[29% abnormal Rab11 accumulation (n514), Fig. 5D; 42%
abnormal Nuf accumulation (n512), Fig. 5E; 37% abnormal Clh
accumulation (n511), Fig. 5F].
In wild-type male meiotic cells, we observed the accumulationof lipid droplets (Fig. 5G–I). Membranous intracellular structures
known as astral membranes that are consisted of ER sheets andenriched in several ER markers are closely associated with astral
microtubules in early embryos (Bobinnec et al., 2003) as well asin primary spermatocytes (Fig. 7A; supplementary material Fig.

Effect of COPI depletion on the formation of central
spindle microtubules

To elucidate the mechanism of disruption of central spindles andastral microtubules in COPI-depleted cells, we investigatedthe microtubule structures. In wild-type metaphase I cells, weobserved large umbrella-shaped astral microtubules emanatingfrom each spindle pole (Fig. 3B; supplementary material Fig. S5).
The central spindles comprise bundles of interdigitatedmicrotubules formed between the separating homologous
chromosomes, and play a key role in contractile ring positioning[Fig. 6A (n.50) and 6C (n526); see supplementary material
Movies 2, 3] (Goldberg et al., 1998; Inoue et al., 2004). We
occasionally observed abnormal cells, in which the direction of
microtubule elongation or orientation was perturbed [42%abnormal microtubule formation (n552); Fig. 6B].
During anaphase, knockdown cells often lack robust central
spindle microtubules. Using a time-lapse experiment, weinvestigated microtubule structures in the COPI-depleted
spermatocytes (Fig. 6D; supplementary material Movies 4, 5).
Following breakdown of the nuclear membrane, markedly fewer
astral or spindle microtubules were constructed, although the
bipolar spindle structure was initially formed. Following
anaphase, the spindle microtubule structures elongated in an
aberrant direction. The interdigitated spindle microtubules from
opposite poles failed to overlap around their plus termini. Thesemicrotubule structures became bent and eventually degraded. At
80 min after onset, COPI-depleted cells exhibited no central
spindles or astral microtubules (Fig. 6D). The primary
spermatocytes did not elongate along the spindle axis after
anaphase, and no cleavage furrow ingression occurred.

Journal of Cell Science

Drosophila male meiosis requires COPI

3655

Fig. 5. COPI depletion results in failure to accumulate vesicle components at the midzone of male meiotic cells. (A–F) Immunolocalization of three vesiclerelated proteins (Rab11, Nuf and clathrin), which are essential for membrane trafficking in cytokinesis. Wild-type (A–C) and aCop knockdown(D–F) spermatocytes were examined. DNA is shown in blue, microtubules in green and immunofluorescence of the three proteins in red. Note that each protein
accumulated at the midzone of early telophase I cells (filled arrowheads) and around the centrosomes. COPI-depleted cells failed to accumulate each protein at the
midzone (open arrowheads). (G–L) Intracellular distribution of lipid droplets in living primary spermatocytes stained with Bodipy 493/503. In wild-type cells, theBodipy fluorescence can be seen on the ER-based astral membranes, and at the midzone of cells (arrowheads) during metaphase (G) to anaphase (H). The
accumulation on ribbon-like structures corresponding to ER-based structures is prominent at telophase; the midzone of the plasma membrane is budding inside, so
as to contact the cellular pool of lipid droplets before cytokinesis (I). In COPI-depleted cells, central accumulation at metaphase to anaphase is not present(J,K); budding of the plasma membrane towards the internal lipid storage is initiated, but membrane ingression fails to contact the storage organelles (openarrowhead in L). Scale bars: 10 mm.

Next, we examined the formation of the anillin rings (known
as pre-contractile rings) in the knockdown spermatocytes(Fig. 6E,F). The formation of anillin rings was frequently
incomplete (Fig. 6F) or absent (36% abnormal ring formation;n536). Taken together, our results suggest that COPI depletion
affects the microtubule structures essential for cytokinesis.
Influence of COPI on central spindle microtubules,
through the formation of ER-based cellular structures

To understand the mechanism by which COPI depletion results inthe formation of abnormal microtubule structures, we investigatedER-based cellular structures. COPI mediates membrane trafficking
between the ER and the Golgi apparatus. In Drosophila meiotic
spermatocytes, the ER, recycling endosomes, and lipid droplets are
frequently associated with the astral membranes and spindleenvelopes (Bobinnec et al., 2003; Dorogova et al., 2009; Fuller,
1993; Tates, 1971). Our time-lapse experimental data confirmed
the overlapping of astral microtubules with ER-based astral
membranes during meiotic division (Fig. 7A). Furthermore, theRtnl1–GFP signal overlapped with the phase-dense intracellular
structures observed using phase-contrast microscope. The astral
membranes at the spindle pole periphery were covered by astral
microtubules, thereby supporting the microtubule structures frominside. The central spindle microtubules were contiguous with thespindle envelope surrounding the nuclear space in whichchromosome segregation occurs; some overlap with the spindleenvelope was evident [similar results were obtained(n518), Fig. 7A; see supplementary material Movies 6, 7, 8;
supplementary material Fig. S7].
We subsequently obtained genetic evidence for a close
relationship between ER-based and microtubule structures inspermatocytes. A dominant mutation for a testis-specific tubulin,bTub85DD, inhibits microtubule polymerization (Kemphues et al.,1980). We observed the disruption of ER-based astral membranes
and spindle envelopes in the bTub85DD/+ cells, but nodistribution of COPI vesicles around the centrosomes [63%distinctly abnormal ER-based structures (n554), supplementarymaterial Fig. S8C]. Our findings suggest that ER-based andmicrotubule structures interact considerably during male meiotic
divisions.
Using a time-lapse experiment, we investigated the way inwhich aberrant ER-based structures are constructed in COPIdepleted cells (39% distinctly abnormal ER-based structures(n518; Fig. 7B; supplementary material Movies 9, 10, 11). In
normal spermatocytes, the ER formed concentric circles on theoutside of the nuclear membrane at prophase. The ER network
consisted of multi-layers of membranous sheets, visible as phasedense structures around the nuclear space. The reticular ER
structure was elongated and transformed into multiple layers ofmembranes (known as spindle envelopes) surrounding the3656

Journal of Cell Science 125 (15)Journal of Cell Sciencestructures. Interestingly, the disorganized microtubules overlappedwith the aberrant ER-based spindle envelopes visualized as phasedense structures (arrowheads in Fig. 7B). Moreover, no cleavage
furrow ingression occurred.
Taken together, our results suggest that COPI depletion
influences the formation of ER-based spindle envelopes, which
mutually interact with the central spindle microtubules essential
for cytokinesis.

Fig. 6. Abnormal meiotic microtubule structures required forcytokinesis, and loss of contractile rings, in COPI-depleted
spermatocytes. (A,B) Fixed meiotic cells from wild-type (A) or aCopdepleted testes (B), Wild-type spermatocytes exhibit robust microtubule
bundles, known as central spindle microtubules, in the middle of anaphasecells. Microtubules are visualized in green; DNA is shown in blue. (C) Timelapse observation of meiotic microtubule structures, and phase-contrast
images of living wild-type spermatocytes. Microtubules were visualized byexpression of GFP–tubulin. The time-lapse images were continuously
collected from anaphase I, in which interior microtubules emanating fromeach spindle pole made contact at the equator (t50 min), to the end ofmeiosis I (t540 min). (D) Time-lapse observation of COPI-depleted
spermatocytes. (E,F) A contractile ring (visualized in red by anillin
immunostaining) is evident in telophase I cells from wild-type males (E) andaCop knockdown males (F). Microtubules are shown in green, and DNA inblue. In the knockdown spermatocytes, elongation of microtubules or theirorientation seems to be disturbed. In anaphase-I-like cells, the central spindle
microtubules are missing, whereas in non-constricted cells within a telophasecyst, a disconnected contractile ring is formed. Scale bars: 10 mm.

nuclear space. In COPI-depleted cells, the ER-based structure
was affected (Fig. 7B; supplementary material Fig. S8C).
Initially, the astral membranes and spindle envelopes wereindistinguishable from those of wild-type cells. However, as thecells elongated at anaphase, the spindle envelopes became curved
in the middle and disconnected (arrowheads in Fig. 7B;
supplementary material Fig. S8).
Simultaneous observation confirmed the interaction of
microtubules and ER-based structures (Fig. 7A,B). In COPIdepleted cells, microtubules exhibited aberrant organization(Fig. 7B,C). At anaphase, spindle microtubules and ER-based
structures aligned side by side. In normal cells, characteristic
microtubule structures (termed exterior central spindles) areconstructed alongside the spindle envelope (Inoue et al., 2004). In
COPI-depleted cells, the orientation of the elongating central spindle
was lost, resulting in the generation of disorganized microtubule

Discussion
In the present study, our initial RNAi screen identified several
COPI subunits and assembly regulators as essential factors forcytokinesis in Drosophila male meiosis. We subsequently
elucidated the cellular function of COPI during Drosophila malemeiosis. We observed the localization of COPI in the ER–Golgi
intermediate compartment of tER–Golgi units scatteredthroughout the spermatocyte cytoplasm. Prior to metaphase,
COPI-containing vesicles assemble at the spindle pole peripherythrough a poleward movement, mediated by astral microtubules
and minus-ended motor dynein. At the end of each meiotic
division, the vesicles are equally partitioned between two daughter
cells. Our findings strongly suggest that spermatocytes possess aregulatory mechanism, to fulfill equal inheritance of several typesof membrane vesicles. Testis-specific knockdown of COPI, or
mutations of the cCOP gene, resulted in defective cytokinesis
during male meiosis. COPI depletion disrupted the accumulationof vesicle components, such as cleavage furrow proteins and lipid
droplets, at the cleavage furrow zone. Furthermore, we observed
aberrant central spindle microtubules. In COPI-depleted cells, theastral membrane and spindle envelope (associated with astral andspindle microtubules, respectively) were severely disrupted. We
propose that these ER-based structures are required as a structural
foundation, to facilitate the formation of meiotic microtubule
structures. Thus, we believe that COPI plays an important role inDrosophila male meiosis, not only through vesicle transport to thecleavage furrow region, but also via the formation of intracellular
structures.
Equal partitioning of membrane vesicles during
Drosophila male meiosis

Giansanti et al. reported that Golgi-derived vesicles wereconcentrated around each spindle pole and excluded from theequatorial region (Giansanti et al., 2007). In gio or fwd mutants,
which encode vesicle transport factors, these vesicles continued
to be accumulated at the equator. The authors concluded that
accumulation of such vesicles was the consequence of failed
incorporation into the membrane at the invaginating cleavage
furrow. However, this phenotype was not observed in othercytokinesis mutants, such as fws and pbl, which encode factors
for Golgi structure and function, and cleavage furrow formation,
respectively (Farkas et al., 2003; Giansanti et al., 2006).
Although Golgi stack components are required for Drosophila
cytokinesis, evidence for the direct fusion of Golgi-derived
vesicles with the furrow membrane remains elusive (Albertson
et al., 2008; Farkas et al., 2003; Xu et al., 2002). Our present
findings indicate the existence of a transport system, whichmediates equal partitioning not only of COPI vesicles, but also ofvesicles containing Lva (cis-Golgi components), during malemeiosis. Thus, we believe that abnormal distribution of Golgiderived vesicles, and of other membranous organelles, resultsfrom the perturbation of vesicle transport.

Journal of Cell Science

Drosophila male meiosis requires COPI

3657

Fig. 7. ER-based intracellular structures associated with astral or spindle microtubules are affected in COPI-depleted spermatocytes. (A) Time-lapse
observation of microtubules (second row) and ER-based structures (third row) in primary spermatocytes expressing mRFP–tubulin and GFP–Rtnl1 (an ERlocalizing enzyme). In merged images (first row), tubulin and Rtnl1 are shown in red and green, respectively. Simultaneous observation by phase-contrast
microscopy is shown in the fourth row (PC). The time-lapse images were continuously collected from mid-anaphase I (t50 min) to the end of meiosis I. Note theassociation of astral and central spindle microtubules with astral membranes and spindle envelopes, respectively. The ER-based structures labeled by GFP–Rtnl1
overlapped with phase-dense structures. (B) Time-lapse observation of COPI-depleted spermatocytes. Bundles of central spindle microtubules were initiallyconstructed, but overlapping microtubules were rapidly degraded, and disappeared by the end of the experiment. Spindle envelopes were less abundant, andbecame malformed and disappeared, in line with the degradation of microtubule structures (arrowheads). Scale bars: 10 mm.

On completion of meiosis during Drosophila spermatogenesis,
considerable cell elongation takes place synchronously in 64
spermatids within a cyst (Fuller, 1993; Inoue et al., 2011). It is
crucial for each spermatid to obtain sufficient numbers ofvesicles. Within spermatids, COPI and other Golgi components
comprise a single large Golgi apparatus, called an acroblast(Farkas et al., 2003). A mammalian counterpart known as theacrosome precursor (Moreno et al., 2000) is required for fusion
between the sperm and oval membranes. Thus, it is likely that
Drosophila spermatocytes possess a mechanism to ensure equal
partitioning of vesicles during male meiosis.

anaphase initiation (Wei and Seemann, 2009). Moreover, thevesicles are transferred by the mitotic spindles during mitosis.
However, it is difficult to distinguish between spindles and asters.
By contrast, in Drosophila spermatocytes, the spindle microtubules
are formed within a nuclear space, surrounded by a multilayer ofparafusorial membranes (Fuller, 1993; Tates, 1971). Drosophila
spermatocytes possess particularly long astral microtubules, and it
is likely that these play an important role in the equal inheritance of
membrane vesicles during male meiosis.

Dependence of COPI vesicle distribution on astral
microtubules and minus-ended motor dynein

In the present study, we have indicated that not only COPI
subunits, but also Arf1, which is a small GTPase required for
COPI assembly are essential for correct execution of cytokinesis
during Drosophila male meiosis. By contrast, in mammaliancultured cells, inactivation of Arf1 is necessary for COPI andclathrin disassembly, which normally occur prior to the initiationof mitosis (Altan-Bonnet et al., 2003). The inhibition of COPI
disassembly causes defects not only in chromosome segregation,
but also in cleavage furrow ingression. Thus, inactivation ofvesicle transport complexes is believed to be indispensable for
mitotic entry. However, the meiotic phenotype of the Drosophila
knockdown cells indicates that the COPI complex plays an activerole in the progression of meiotic divisions. The significance of
COPI in cell division may be dependent on cell types. For smaller
cells to initiate cell division, it is important to shut downcytoplasmic activities, such as protein synthesis and vesicletransport (Hernandez-Verdun, 2011; Hernandez-Verdun et al.,2002). By contrast, in Drosophila spermatocytes, membranous
cellular structures, such as multi-layers of parafusorial

Our present findings indicate that vesicle transport towards thespindle pole periphery is mediated by minus-ended motor dynein.
Dynein was previously reported to form complexes with Lvacontaining vesicles, which may correspond to Golgi stacks(Papoulas et al., 2005). It is reasonable to speculate that other
Golgi-derived vesicles are transported along astral microtubules by
motor dynein. In budding yeast, the actin cytoskeleton plays anessential role in Golgi transport during mitosis (Arai et al., 2008).
We failed to observe co-localization of COPI vesicles with actin
networks in male meiotic cells (D.K. and Y.H.I., unpublished data).
It is likely that microtubule structures play a key role in theinheritance of vesicles during male meiosis. Ordered vesicle
inheritance in mammalian male meiotic divisions has not been
demonstrated. In mammalian cultured cells, the Golgi ribbon is
fragmented into smaller pieces by the onset of mitosis (Rabouille
and Jolo, 2003; Shima et al., 1998). Wei and Seemann reported that
COPI vesicles accumulate around the centrosomes prior toIndispensability of Arf1-regulated, COPI-mediatedmembrane trafficking during Drosophila male meiosis

3658

Journal of Cell Science 125 (15)membranes, are well developed. The ER-based structures on
which COPI function depends undergo a massive alteration
during meiotic divisions (Fig. 7A). Spermatocytes are muchlarger than other somatic cells, and may need to maintain higher
COPI activity, not only at interphase, but also during cell
division. The involvement of membrane trafficking components
in cell division was recently reported. The membranous spindle
matrix, which has an ER feature, influences spindle morphology
during partially open mitosis in Drosophila early embryos, andalso during open mitosis in vertebrate cells (Zheng, 2010; Liu andZheng, 2009; Civelekoglu-Scholey et al., 2010). The endocytic
adaptor protein, epsin I, was also shown to regulate mitotic
membrane organization and spindle morphology (Liu and Zheng,2009).

to the cleavage furrow zone. Nevertheless, we cannot exclude thepossibility that the failure of vesicle component accumulation in
COPI-depleted cells is a consequence, rather than a cause ofcytokinesis defects. Detailed observations of cleavage furrow
proteins in living meiotic cells are required to clarify this issue.
COPI is required for the construction of ER-based spindleenvelopes, which appear mutually to interact with the central
spindle microtubules. It is well known that these microtubule
structures and the contractile ring are interdependent (Giansantiet al., 1998). Thus, it is possible to speculate that COPI depletion
eventually influences contractile ring formation, which isessential for the initiation of cytokinesis.
Materials and Methods
Drosophila stocks

Journal of Cell Science

Role of ER-based structures as a structural foundation toensure correct formation of the cell division apparatus

Our present findings indicate that meiotic microtubules and ERbased structures mutually interact. Furthermore, the ER-based
astral membranes and spindle envelopes may serve as structural
foundations, to ensure the correct formation of astral and spindle
microtubules, respectively. Rebollo and González observed thatcolchicine treatment of primary spermatocytes undergoing malemeiosis influenced phase-dense structures corresponding to ERbased structures (Rebollo and González, 2000). Barbosa et al.
reported that mutants of dd4 genes encoding spindle pole factors
led to abnormalities in spindle microtubules and phase-dense
structures during male meiosis (Barbosa et al., 2000). Further
studies using live analysis of mutations affecting additional
microtubule structures are required to verify our hypothesis
concerning the mutual interaction of meiotic microtubules andER-based structures. The role of ER structures in microtubule
elongation may be compared to that of a splint in the regenerationof broken bones. In the COPI knockdown cells, the microtubules
may have lacked support for elongation in the correct orientation.
Some microtubules can initiate elongation independently ofcentrosomes (Rebollo et al., 2004). In human, and also
Drosophila cells, these noncentrosomal microtubules may be
dependent on seed proteins for elongation from the surface ofother microtubules (Rogers et al., 2008; Uehara et al., 2009).
Thus, in Drosophila spermatocytes, advanced ER structures may
be useful for facilitating continuous and straight elongation of theparticularly long astral microtubules.
The microtubule assembly checkpoint in spermatocytes
operates less strictly than does the mitotic cycle (Rebollo andGonzález, 2000). Moreover, Drosophila spermatocytes arenotable for their well-developed membranous structures(Stafstrom and Staehelin, 1984; Tates, 1971). Thus, the ER
structure may facilitate correct formation of the cell divisionapparatus, to compensate for the incomplete checkpoint system.
In summary, we have demonstrated that COPI depletion resultsin the failure of cytokinesis, through disrupted accumulation ofvesicles at the cleavage furrow region. Mutations for a
Drosophila golgin ortholog, Lva (which mediates Golgi
transport), resulted in a similar cytokinesis phenotype (Sisson
et al., 2000). The delivery of membrane components to thecleavage furrow region is thought to involve two different
pathways, the secretory pathway and the endocytic pathway.
COPI is related to Golgi functions, and therefore COPI-mediatedmembrane trafficking may be required for the delivery of vesicle
components required for cytokinesis, via the secretory pathway,

y w, or btubulin–GFP stocks that has been previously described (Inoue et al., 2004)
were used as a wild-type control for cytological and time-lapse studies. Stocks
containing UAS-RNAi constructs of 52 genes, including genes for the seven COPI
subunits (aCop, bCop, b9Cop, cCop, dCop, eCop, and fCop), five arf genes
encoding COPI assembly factors (arf79F, arf102F, arf51F, arf72A, and arf84F),
and 40 other membrane trafficking-related factors, were obtained from the Vienna
Drosophila RNAi Center. We used UAS-dir2; bam-Gal4::VP16 (a gift from T.
Noguchi) or nanos-Gal4::VP16 for spermatocyte-specific gene induction or
knockdown. The following stocks were obtained from the Bloomington Stock
Center: P{sqh-EYFP-Golgi}, P{sqh-EYFP-ER}, nanos-Gal4::VP16, P{GAL4arm.S}4a P{GAL4-arm.S}4b, and P{SUPor-P}cCopKG06383. Protein trap stocks
expressing GFP–Rtnl1 (#G00071) and GFP–PDI (#G00198III) were obtained from
L. Cooley; cCop{XP}d06498 was supplied by the Exelixis Collection at Harvard;
asl1 and asl2 were gifts from S. Bonaccorsi (Rebollo et al., 2004); and orbit7 was as
described previously (Inoue et al., 2004).
aCOP antibody and western blot analysis

A 1.3-kb BamHI–EcoRI fragment of Drosophila aCOP cDNA was inserted inframe into expression vector pET24b (Novagen, Madison, WI, USA). The
resulting plasmid expresses a fusion protein of a polypeptide corresponding toamino acids 548–972 with a stretch of 14 amino acids at the NH3 terminus and astretch of 18 amino acids at the COOH terminus as linkers after the COOH 66His
tag. The recombinant His–aCOP protein was purified by Ni-NTA Spin Kit(Qiagen, Valencia, CA, USA). Antiserum was prepared by injecting a guinea pig.
Western blots were incubated with the antiserum diluted 1:5000, followed byincubation with HRP-conjugated anti-guinea pig IgG.
Live cell imaging of primary spermatocytes

To observe cytokinesis in living primary spermatocytes more easily, we added
some modifications to the protocol described previously (Inoue et al., 2004). To
examine dynamics of COPI in male meiosis, flies expressing mRFP–cCOP (a giftfrom N. Grieder) were used. Testes from the adult flies were dissected and cells
were laid out under mineral oil (Trinity Biotech, Bray, Ireland) in open chambers
surrounded by double-faced tape on clean glass coverslips without any pressure.
Using this protocol, we have succeeded in continuous observation of primary
spermatocytes undergoing proper spindle formation and cytokinesis for at least anhour. For a drug treatment, we carried out short term in vitro culture of primary
spermatocytes according to methods described previously (Robinett et al., 2009)
with minor modification. A testis complex attached with accessory gland,ejaculatory duct and pomp were dissected from adult males. A living testis
complex in which ejaculatory pomp is actively contracting was selected andtransferred into a culture medium consisting of modified M3 medium withoutbicarbonates (Sigma-Aldrich, St Louis, MO, USA) containing 10% fetal calf
serum (Sigma-Aldrich) and 50% male cell extracts. To inhibit COPI assembly,
aCOPI inhibitor, brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) or Exo1
(Sigma-Aldrich) was directly added to the culture medium. The testis was
incubated in the culture medium for 14 hours before isolation of spermatocytes atroom temperature. For observation of lipid droplets, Bodipy 493/503 (4,4-difluoro1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacence; Molecular Probes, Eugene,
OR, USA) or Nile red (Wako, Osaka, Japan) was used. Time-lapse imaging was
performed on an Olympus IX81 fluorescence microscope (Olympus, Tokyo,
Japan) outfitted with excitation, emission filter wheels (Olympus, Tokyo, Japan).
Cells were imaged with a 406 lens. At each 30 sec time interval, nearsimultaneous GFP and RFP fluorescence images were captured with a CCD
camera (Hamamatsu Photonics, Hamamatsu, Japan). Image acquisition was
controlled through the Metamorph software package running on a PC. Testissquashes to evaluate onion-stage spermatids were made using previous protocols(Inoue et al., 2004) and viewed by phase-contrast microscopy.

Drosophila male meiosis requires COPI
Immunofluorescence

Testis cells were fixed according to the method of Inoue and colleagues (Inoueet al., 2004). For immunostaining, anti-aCOP antibody was used at a 1:500
dilution. The following primary antibodies were used: anti-GM130 (Abcam,
Cambridge, MA, USA), anti–cCOP (a gift from Wieland) (Jayaram et al., 2008),
anti-centrosomin (T. Kaufman), anti-anillin (D. Glover), anti-Lva (W. Sullivan)
and anti-Clathrin (S. Kametaka). For anti-Rab11 (R. Choen) staining was carriedout by a method described previously (Giansanti et al., 2007). Microtubules werevisualized by immunostaining with anti-a-tubulin (DM1A: Sigma-Aldrich) or
expression of GFP–b-tubulin (Inoue et al., 2004). ER was visualized by expressionof GFP–Rtnl1 residing predominantly in the intracellular structure. All secondary
antibodies and DNA stains were commercially obtained. Images were processed
and merged in pseudocolor using MetaMorph version 7.6 (Molecular Devices,
Sunnyvale, CA, USA).

Acknowledgements

We would like to thank S. Taketani and S. Miyata for technicaladvice on establishing antibodies. We are grateful to N. Grieder, S.
Bonaccorsi, L. Cooley and DGRC, VDRC, BSC, for providing fly
stocks, and to F. Wieland, D. Glover, S. Kametaka, R. Cohen, andW. Sullivan for antibodies.
Funding

Journal of Cell Science

This work was supported by Grants-in-Aid for Scientific Research on
Priority Area and for Scientific Research (C) [grant number
23570004 to Y.H.I.]
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.103317/-/DC1

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